Antibody Purification

ABSTRACT

Methods for the purification of antibodies are provided. Purification methods provided involve the use of hydroxyapatite resin (HA) to separate an antibody of interest from one or more impurities. The impurity may be a clipped antibody that comprises a cleaved peptide bind in the VH domain.

This application claims the benefit of U.S. Provisional Application No.62/635,943 filed on Feb. 27, 2018, the contents of which is herebyincorporated by reference in its entirety.

FIELD

The present invention relates to methods for purifying antibodies fromimpurities, such as antibody degradation products. Purification methodsdisclosed herein involve the use of hydroxyapatite resins.

BACKGROUND

Antibodies are important biologic molecules for medical, diagnostic,industrial, and other uses. While many methods and reagents areavailable for the recombinant production of antibodies, due to, forexample, the size and molecular complexity of antibodies, it frequentlyremains difficult to efficiently produce and purify a recombinantantibody of interest, particularly at large/industrial scale productionlevels.

For example, during the production of a recombinant antibody ofinterest, at times, a degradation product related to the antibody ofinterest may arise; the degradation product is an unwanted impurity.This degradation product may have some molecular properties that arevery similar to the antibody of interest (e.g. identical or almostidentical amino acid sequences or mass). Because of the molecularsimilarities between the intact antibody of interest and the degradedversion of the antibody, it may be very difficult to effectivelyseparate the intact antibody from the degraded antibody.

Accordingly, there is a need for new and improved methods for thepurification of antibodies from impurities.

SUMMARY

Provided herein are methods for purifying an antibody of interest fromone or more impurities.

In some embodiments, provided herein is a method of purifying anantibody comprising: A) loading an antibody preparation in a load bufferonto a hydroxyapatite (HA) resin, wherein: the antibody preparationcomprises: I) an intact antibody of interest and II) a clipped versionof the antibody of interest, wherein the clipped version of the antibodyof interest is a degradation production from the intact antibody ofinterest, and has a mass that is less than 10% different than the massof the intact antibody of interest; and B) eluting the intact antibodyof interest from the HA resin with an elution buffer.

In some embodiments, provided herein is a method of purifying abispecific antibody comprising: A) loading an antibody preparation in aload buffer onto a hydroxyapatite (HA) resin, wherein: the antibodypreparation comprises: I) an intact bispecific antibody of interest; andII) at least one impurity species, wherein the impurity species areselected from the group consisting of: a) a clipped version of thebispecific antibody of interest, wherein the clipped version of thebispecific antibody of interest is a degradation production from theintact bispecific antibody of interest, and has a mass that is less than10% different than the mass of the intact bispecific antibody ofinterest; b) a first parent antibody, wherein the first parent antibodyis a monospecific antibody having the same antigen specificity as afirst arm of the intact bispecific antibody; c) a second parentantibody, wherein the second parent antibody is a monospecific antibodyhaving the same antigen specificity as a second arm of the intactbispecific antibody; and d) high molecular mass species (HMMS); and B)eluting the intact bispecific antibody of interest from the HA resinwith an elution buffer.

In some embodiments, provided herein is a method of purifying abispecific antibody comprising: A) loading an antibody preparation in aload buffer onto a hydroxyapatite (HA) resin, wherein: I) the antibodypreparation comprises: a) an intact bispecific antibody of interest andb) a clipped version of the bispecific antibody of interest, wherein theclipped version of the antibody of interest is a degradation productionfrom the intact bispecific antibody of interest, and has a mass that isless than 10% different than the mass of the intact bispecific antibodyof interest; and II) the ratio of molecules of the clipped bispecificantibody to molecules of the intact bispecific antibody in the antibodypreparation is between at least 1:50 and no greater than 1:5; B) elutingthe intact bispecific antibody from the HA resin with an elution buffer.In some embodiments, the method further comprises the step of C)collecting a purified fraction eluted from the HA resin, wherein thepurified fraction comprises the intact bispecific antibody.

In some embodiments, provided herein is a method of purifying anantibody comprising: A) loading an antibody preparation in a load bufferonto a hydroxyapatite (HA) resin, wherein: I) the antibody preparationcomprises: a) an intact antibody of interest and b) a clipped version ofthe antibody of interest, wherein the clipped version of the antibody ofinterest is a degradation production from the intact antibody ofinterest, and has a mass that is less than 10% different than the massof the intact antibody of interest; and II) the clipped version of theantibody comprises at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%,15%, or 20% of the antibody preparation by mass; B) eluting the intactantibody from the HA resin with an elution buffer, and C) collecting apurified fraction eluted from the HA resin, wherein the purifiedfraction comprises the intact antibody, and contains less than 1%, 2%,3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, or 20% by mass clipped antibody,wherein the purified fraction contains a lower % by mass clippedantibody than the antibody preparation.

In some embodiments, in a method provided herein involving eluting anantibody of interest from an HA resin with an elution buffer, theelution buffer comprises an ion. Optionally, the concentration of theion in the buffer is increased during the elution. Optionally, theconcentration of the ion in the buffer around the HA resin is increasedduring the elution.

In some embodiments, in a method provided herein involving an antibody,the antibody is a heterodimeric bispecific antibody.

In some embodiments, in a method provided herein involving collecting apurified fraction eluted from the HA resin, wherein the purifiedfraction comprises the intact antibody of interest, the purifiedfraction comprises at least 80%, 90%, 95%, 96%, 97%, 98%, or 99% by massintact antibody of interest.

In some embodiments, in a method provided herein involving a purifiedfraction comprising an intact bispecific antibody and a clippedbispecific antibody, the ratio of clipped bispecific antibody moleculesto intact bispecific antibody molecules in the purified fraction is nogreater than 1:400, 1:200, 1:100, or 1:50.

In some embodiments, in a method provided herein involving an antibodyof interest that is an anti-CD3 antibody or that is a bispecificantibody that contains an anti-CD3 arm, the antibody comprises at leastone of the following: i) a VH region comprising an amino acid sequenceas shown in SEQ ID NO: 1; ii) a heavy chain comprising an amino acidsequence as shown in SEQ ID NO: 2; iii) a VH region comprising an aminoacid sequence as shown in SEQ ID NO: 1 and a VL region comprising anamino acid sequence as shown in SEQ ID NO: 3; or iv) a heavy chaincomprising an amino acid sequence as shown in SEQ ID NO: 2 and a lightchain comprising an amino acid sequence as shown in SEQ ID NO: 4.

In some embodiments, in a method provided herein involving a bispecificantibody, the bispecific antibody is: i) an anti-BCMA/anti-CD3bispecific antibody comprising an anti-BCMA arm and an anti-CD3 arm, orii) an anti-FLT3/anti-CD3 bispecific antibody comprising an anti-FLT3arm and an anti-CD3 arm.

In some embodiments, in a method provided herein involving a bispecificantibody comprising an anti-BCMA arm, the anti-BCMA arm comprises atleast one of the following: i) a VH region comprising an amino acidsequence as shown in SEQ ID NO: 5; ii) a heavy chain comprising an aminoacid sequence as shown in SEQ ID NO: 6; iii) a VH region comprising anamino acid sequence as shown in SEQ ID NO: 5 and a VL region comprisingan amino acid sequence as shown in SEQ ID NO: 7; or iv) a heavy chaincomprising an amino acid sequence as shown in SEQ ID NO: 6 and a lightchain comprising an amino acid sequence as shown in SEQ ID NO: 8.

In some embodiments, in a method provided herein involving a bispecificantibody comprising an anti-FLT3 arm, the anti-FLT3 arm comprises atleast one of the following: i) a VH region comprising an amino acidsequence as shown in SEQ ID NO: 9; ii) a heavy chain comprising an aminoacid sequence as shown in SEQ ID NO: 10; iii) a VH region comprising anamino acid sequence as shown in SEQ ID NO: 9 and a VL region comprisingan amino acid sequence as shown in SEQ ID NO: 11; or iv) a heavy chaincomprising an amino acid sequence as shown in SEQ ID NO: 10 and a lightchain comprising an amino acid sequence as shown in SEQ ID NO: 12.

In some embodiments, in a method provided herein involving loading anantibody preparation onto an HA resin, the antibody preparation isloaded onto the HA resin to a density on the resin of between 2, 3, 4,or 5 g/L and 8, 9, 10, 12, 15, or 20 g/L.

In some embodiments, in a method provided herein involving loading anantibody preparation onto an HA resin, at least 1, 5, 10, 50, 100, 500,1000, or 5000 grams of antibody preparation is loaded onto the HA resin.

In some embodiments, in a method provided herein involving an antibodypreparation, the antibody preparation comprises at least 50%, 60%, 70%,or 80% but less than 90% 95%, 97%, 98%, or 99% by mass intact antibodyof interest.

In some embodiments, in a method provided herein involving a clippedantibody, the clipped antibody has a mass that is less than 0.1%, 0.5%,1%, or 2% different than the mass of the intact antibody. In someembodiments, in a method provided herein involving a clipped antibody,the clipped antibody has a mass that is between about 5 and 100 Daltonsgreater than the mass of the intact antibody. In some embodiments, in amethod provided herein involving a clipped antibody, the clippedantibody has a mass that is about 18 Daltons greater than the mass ofthe intact antibody.

In some embodiments, in a method provided herein involving a clippedantibody, the clipped antibody has a cleaved peptide bond in apolypeptide chain of the antibody, and wherein the cleaved peptide bondis in a heavy chain of the antibody. In some embodiments, in a methodprovided herein involving a clipped antibody, the clipped antibody has acleaved peptide bond in a polypeptide chain of the antibody, and whereinthe cleaved peptide bond is in a light chain of the antibody.

In some embodiments, in a method provided herein involving a clippedantibody, the clipped antibody contains the same number of amino acidsand the same amino acid sequences as the intact antibody. Alternatively,in some embodiments, the clipped antibody contains a different number ofamino acids as the intact antibody.

In some embodiments, in a method provided herein involving an antibodyof interest that comprises a VH and VL domain which specifically bind toCD3, a corresponding clipped antibody comprises a cleaved peptide bondin the VH domain that specifically binds CD3.

In some embodiments, in a method provided herein involving an HA resin,the HA resin is ceramic hydroxyapatite (cHA) resin.

In some embodiments, in a method provided herein, an HA resin is washedwith a wash buffer comprising phosphate ions after loading the antibodypreparation onto the HA resin but prior to eluting the intact bispecificantibody from the resin. Optionally, the wash buffer comprises phosphateions at concentration between about 5, 10, 15, 20, and 30, 40, or 50 mM.

In some embodiments, in a method provided herein involving an elutionbuffer containing an ion, the ion is phosphate. In some embodiments, theconcentration of phosphate ion during elution may increase from about30, 40, or 50 mM to about 60, 70, 80, 100, 150, or 200 mM.

In some embodiments, in a method provided herein the pH of at least oneof the load buffer, wash buffer, and elution buffer is at or betweenabout pH 7.0 and 8.0.

In some embodiments, in a method provided herein involving an antibodypreparation, the antibody preparation contains proteins that werepreviously loaded onto and eluted from at least one of: i) a protein Aresin and ii) an ion exchange resin. Optionally, the antibodypreparation contains proteins that were previously loaded onto andeluted from both of: i) a protein A resin and ii) an ion exchange resin.

In some embodiments, the antibody prepared using the method as describedherein is isolated and/or purified for use as or in the preparation ofpharmaceuticals.

In some embodiments, provided is an antibody purified using the methodsas described herein.

BRIEF DESCRIPTION OF THE FIGURES/DRAWINGS

FIG. 1 depicts a schematic representation of a method of preparing abispecific antibody that may be purified according to methods providedherein.

FIG. 2 depicts a schematic representation of an exemplary i) intactbispecific antibody (left side panel) and ii) clipped version of thebispecific antibody (right side panel), in which the clipped bispecificantibody is an impurity that may be present in an antibody preparationwith the intact bispecific antibody.

FIG. 3 depicts a chromatogram showing the separation of ananti-BCMA/anti-CD3 bispecific antibody of interest (“POI”) from multipledifferent impurities via elution from an HA resin.

FIG. 4 depicts a graph showing the relative amounts of different proteinspecies (including the antibody of interest and various impurities) indifferent fractions eluted from an HA resin according to an HAchromatography run as depicted in the chromatogram of FIG. 3.

FIG. 5 depicts a chromatogram showing the separation of ananti-BCMA/anti-CD3 bispecific antibody of interest (“POI”) from multipledifferent impurities via elution from an HA resin.

FIG. 6 depicts a graph showing the relative amounts of different proteinspecies (including the antibody of interest and various impurities) indifferent fractions eluted from an HA resin according to an HAchromatography run as depicted in the chromatogram of FIG. 5, in whichan anti-BCMA/anti-CD3 bispecific antibody of interest is separated frommultiple different impurities via elution from an HA resin

FIG. 7 depicts a graph showing the relative amounts of different proteinspecies (including the antibody of interest and various impurities) indifferent fractions eluted from an HA resin according to an HAchromatography run, in which an anti-FLT3/anti-CD3 bispecific antibodyof interest is separated from multiple different impurities via elutionfrom an HA resin.

DETAILED DESCRIPTION

Provided herein are methods for purifying an antibody of interest fromone or more impurities. Methods provided herein involve the use of ahydroxyapatite resin to separate the antibody of interest fromimpurities. In some embodiments, the antibody of interest is abispecific antibody. In some embodiments, an impurity is an antibodythat is related to the antibody of interest (i.e. it has a similar orthe same amino acid sequence(s) as the antibody of interest), but it ismodified in one or more ways as compared to the antibody of interest,and it has a different mass than the antibody of interest. Optionally,the mass of an antibody impurity species is very similar to the mass ofthe antibody of interest. For example, in some embodiments, the mass ofan antibody impurity species is less than 5%, 2%, 1%, 0.5%, 0.2%, 0.1%,0.05%, 0.02%, or 0.01% different from the mass of the antibody ofinterest. Optionally, methods provided herein may be used for the largescale purification of an antibody of interest from one or moreimpurities.

Definitions

Unless otherwise defined, all terms of art, notations and otherscientific terms or terminology used herein are intended to have themeanings commonly understood by those of skill in the art to which thisinvention pertains. In some cases, terms with commonly understoodmeanings are defined herein for clarity and/or for ready reference, andthe inclusion of such definitions herein should not necessarily beconstrued to represent a substantial difference over what is generallyunderstood in the art.

The following terms, unless otherwise indicated, shall be understood tohave the following meanings:

An “antibody” is an immunoglobulin molecule capable of specific bindingto a target, such as a carbohydrate, polynucleotide, lipid, polypeptide,etc., through at least one antigen recognition site, located in thevariable region of the immunoglobulin molecule. As used herein, the termencompasses not only intact polyclonal or monoclonal antibodies, butalso fragments thereof (such as Fab, Fab′, F(ab′)₂, Fv), single chain(ScFv) and domain antibodies (including, for example, shark and camelidantibodies), diabodies, and fusion proteins comprising an antibody, andany other modified configuration of the immunoglobulin molecule thatcomprises an antigen recognition site. The term “antibody” includesmonospecific, bispecific, and multispecific antibodies. An antibodyincludes an antibody of any class, such as IgG, IgA, or IgM (or subclassthereof), and the antibody need not be of any particular class.Depending on the antibody amino acid sequence of the constant region ofits heavy chains, immunoglobulins can be assigned to different classes.There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, andIgM, and several of these may be further divided into subclasses(isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2. The heavy-chainconstant regions that correspond to the different classes ofimmunoglobulins are called alpha, delta, epsilon, gamma, and mu,respectively. The subunit structures and three-dimensionalconfigurations of different classes of immunoglobulins are well known.

As used herein, the terms “heavy chain”, “light chain”, “variableregion” or “variable domain”, “framework region”, “constant domain”, andthe like, have their ordinary meaning in the immunology art and refer todomains in naturally occurring immunoglobulins and the correspondingdomains of recombinant binding proteins (e.g. humanized antibodies,bispecific antibodies, single chain antibodies, chimeric antibodies,etc.). The basic structural unit of naturally occurring immunoglobulinsis a tetramer having two light chains and two heavy chains, usuallyexpressed as a glycoprotein of about 150,000 Da. The amino-terminal(N-terminal) portion of each chain includes a variable region of about100 to 110 or more amino acids primarily responsible for antigenrecognition. The carboxy-terminal (C-terminal) portion of each chaindefines a constant region. Each light chain is comprised a light chainvariable domain (VL) and a light chain constant domain (CL). Each heavychain is comprised of a heavy chain variable region (VH) and a heavychain constant region, having CH1, hinge, CH2 and CH3 domains. Thevariable regions of an IgG molecule comprise regions ofhypervariability, termed the complementarity determining regions (CDRs),which contain the residues in contact with antigen, and non-CDRsegments, termed framework regains (FR), which generally maintain thestructure and determine the positioning of the CDR loops (althoughcertain framework residues may also contact antigen). Each VH and VLcomprises three CDRs and four FRs, arranged from amino-term nus tocarboxy-terminus in the following structure: n-FR1, CDR1, FR2, CDR2,FR3, CDR3, FR4-c. Immunoglobulin molecules can be of any type (e.g.,IgG, IgE, IgM, IgD, IgA and IgY) and class (e.g., IgGI, IgG2, IgG 3,IgG4, IgA1 and IgA2) or subclass.

A “bispecific” or “dual-specific” is a hybrid antibody having twodifferent antigen binding sites. The two antigen binding sites of abispecific antibody bind to two different epitopes, which may reside onthe same or different protein targets.

An “intact” antibody refers to a recombinant antibody that contains allof the expected peptide bonds and amino acids of the recombinantantibody (i.e. that would be expected based on the nucleic acidsequence(s) encoding the polypeptide(s) of the antibody). In contrast, a“clipped” antibody refers to a version of the corresponding “intact”antibody that is missing at least one peptide bond, as compared to thecorresponding “intact” antibody. References herein to an “antibody ofinterest” generally refer to an intact antibody of interest, unless thecontext clearly dictates otherwise.

Reference to “about” a value or parameter herein includes embodimentsthat are directed to that value or parameter per se, as well as tovalues or parameters that may be as much as 10% below or above thestated numerical value for that parameter. For example, a reference to“about 5 mg” includes 5 mg and also any value between 4.5 mg and 5.5 mg.

Methods

Methods provided herein may be used to purify an antibody of interestaway from one or more impurities. In methods provided herein, anantibody preparation (also referred to herein as a “starting sample”)containing the antibody of interest and one or more impurity moleculesis loaded onto a hydroxyapatite (HA) resin, which binds to the antibodyof interest and optionally one or more impurity molecules. The HA resinis then washed to remove any loosely bound impurities. (In someembodiments, all impurity molecules may flow through and not bind to theHA resin.) Next, the antibody of interest is eluted from the HA resinusing a phosphate elution buffer, which is typically introduced onto theresin via a gradient of increasing phosphate ion concentration. Elutionof the antibody of interest from the HA resin yields a purified samplecontaining the antibody of interest, and fewer (or no) impurities thanwere present with the antibody of interest in the starting sample.During the elution of the antibody of interest from the HA resin, anyimpurity molecules bound to the HA resin may also elute at some pointduring the gradient of increasing phosphate ion concentration. However,the impurity molecules elute from the HA resin under sufficientlydifferent conditions from the conditions of elution of the antibody ofinterest, such that the antibody of interest may be effectivelyseparated from the impurity molecules during the elution process.Additional details about the above method steps and related materialsand steps are provided below.

Hydroxyapatite Resin

Various hydroxyapatite resins are available commercially, and anyavailable form of the material can be used with methods provided herein.Optionally, a hydroxyapatite is in a crystalline form. Optionally, ahydroxyapatite is agglomerated to form particles and sintered at hightemperatures into a stable porous ceramic mass.

In some embodiments, an HA resin provided herein is a ceramichydroxyapatite (cHA) resin. “ceramic hydroxyapatite”/“cHA” refers to aninsoluble hydroxylated calcium phosphate of the formula Ca₁₀(PO₄)₆(OH)₂,which has been sintered at high temperatures into a spherical,macroporous ceramic form. As used herein “ceramic hydroxyapatite”/“cHA”encompasses, but is not limited to, Type I and Type II ceramichydroxyapatite, and also encompasses any suitable particle size, unlessotherwise specified. Typical cHA particle sizes that may be used withmethods provided herein, include, for example, a particle size between1-100 μm or 1-1000 μm in diameter, such as 20 μm, 40 μm or 80 μm.Exemplary cHA resins that may be used with methods provided hereininclude CHT™ Type I and Type II resins (Bio-Rad). Any reference hereinto an “HA resin” or the like encompasses cHA resin.

Typically, in a method provided herein, the HA resin is provided in oneor more chromatography columns. The column properties, such as thecolumn's diameter, length, and packing density can be selected based onvarious factors, including the needs of a particular purificationproject (i.e. the amount of protein to be purified), and factorsrelating to the HA resin to be used in the column, such as the its poresize, particle size, compressibility, load capacity, and dynamic bindingcapacity. In addition, methods provided herein are frequently describedin relation to HA resin in a chromatography column; however, othersuitable related configurations for the resin are not excluded. Also,reference herein to a “HA column”, or the like refers to achromatography column that is packed with a HA resin.

Equilibrating a HA Column Prior to Protein Loading

In some embodiments, prior to loading a sample containing an antibody ofinterest onto an HA column, methods provided herein may comprise a stepof pre-equilibrating the column with one or more equilibrationbuffer(s). The equilibration buffers may be introduced onto the column,for example, to ensure that the HA resin is clean at the start of themethod (i.e. to ensure that the resin does not have impurities alreadybound to the resin) and/or to ensure that the solution surrounding theHA resin is compatible with the sample to be loaded onto the resin.

In some embodiments, an equilibration buffer is a phosphate buffercomprising, for example, sodium phosphate, wherein the concentration ofthe phosphate ions in the buffer is from about 100 to 500 mM. Suchequilibration buffers may also be referred to herein as “high phosphateequilibration buffers” or the like. For example, in an embodiment, ahigh phosphate equilibration buffer may contain about 250 to 450 mMphosphate ions; in other embodiments, it may contain about 200, 250,300, 350, 400, or 450 mM phosphate ions. This equilibration buffercontains a relatively high concentration of phosphate ions in the bufferto elute any contaminants/impurities that are already present on the HAresin (i.e. that are there before the sample containing the protein ofinterest is loaded onto the resin; such impurities might be present, forexample, if the HA resin had been used previously for a purificationmethod, and the resin was not fully cleaned after the previous use). Ahigh phosphate equilibration buffer may have a pH of about 6.0 to 9.0.For example, in an embodiment, a high phosphate equilibration buffer mayhave a pH of about 7.0 to 8.0; in other embodiments, it may have a pH ofabout 7.0, 7.5, or 8.0. In one embodiment, a high phosphateequilibration buffer contains about 400 mM phosphate ions, and has a pHof about 7.5. In some embodiments, a high phosphate equilibration buffermay also be referred to herein as “Equilibration Buffer 1”.

In some embodiments, an equilibration buffer is a phosphate buffercomprising, for example, sodium phosphate, wherein the concentration ofthe phosphate ions in the buffer is from about 1 to 20 mM. Suchequilibration buffers may also be referred to herein as “low phosphateequilibration buffers” or the like. For example, in an embodiment, a lowphosphate equilibration buffer may contain about 1 to 10 mM phosphateions; in other embodiments, it may contain about 1, 2, 3, 4, 5, or 10 mMphosphate ions. This equilibration buffer contains a relatively lowconcentration of phosphate ions in the buffer in order to generateconditions around the HA resin conducive for the protein of interest tobind to the resin. Optionally, a low phosphate equilibration buffer mayadditionally contain HEPES in a concentration from about 1 to 50 mM. Forexample, in an embodiment, a low phosphate equilibration buffer maycontain about 2 to 30 mM HEPES; in other embodiments, it may containabout 5, 10, 15, 20, or 25 mM HEPES. A low phosphate equilibrationbuffer may have a pH of about 6.0 to 9.0. For example, in an embodiment,a low phosphate equilibration buffer may have a pH of about 7.0 to 8.0;in other embodiments, it may have a pH of about 7.0, 7.5, or 8.0. In oneembodiment, a low phosphate equilibration buffer contains about 2 mMphosphate ions, 20 mM HEPES, and has a pH of about 7.5. In someembodiments, a low phosphate equilibration buffer may also be referredto herein as “Equilibration Buffer 2”. Importantly, however, withmethods provided herein, a low phosphate equilibration buffer may beused to pre-equilibrate the resin without the prior use of a highphosphate equilibration buffer during the method.

Loading a HA Column with the Sample Containing the Antibody of Interest

Once a HA column is ready for protein loading, the sample containing theantibody of interest and impurities is loaded onto the HA column. Thebuffer in which the sample loaded onto the HA column may be referred toherein as the “load buffer”. In some embodiments, when a samplecontaining an antibody of interest is initially obtained for use with amethod as provided herein, the sample is already in a suitable loadbuffer for loading the sample onto the HA column. In other embodiments,however, prior to loading a sample onto the HA column, the sample may betreated (e.g. diluted, concentrated, or buffer exchanged) in order tomodify the buffer conditions of the sample, such that the sample will bein a suitable buffer for loading onto the column. For example, withmethods provided herein, a load buffer cannot have a high concentrationof phosphate ions that would impede the binding of the antibody ofinterest to the HA resin. Accordingly, if an initial sample containingthe antibody of interest contains a high concentration of phosphateions, that sample would need to be, for example diluted or bufferexchanged, until the concentration of phosphate ions in the sample isreduced to a suitably low concentration that permits the binding of theantibody of interest to the HA resin.

In some embodiments, a load buffer contains no more than about 10 mMphosphate ions. For example, in some embodiments, a load buffer containsless than about 10 mM, 5 mM, 4 mM, 3 mM, 2 mM, or 1 mM phosphate ions.In some embodiments a load buffer contains 0 mM phosphate ions.Optionally, a load buffer may contain various other salts or buffercomponents (e.g. Tris, glycine). A load buffer may have a pH of about6.0 to 9.0. For example, in an embodiment, a load buffer may have a pHof about 7.0 to 8.0; in other embodiments, it may have a pH of about7.0, 7.5, or 8.0.

In some embodiments, a sample containing the antibody of interest may beloaded onto an HA resin to a density on the resin of at least 1 g/L, 2g/L, 3 g/L, 4 g/L, 5 g/L, 6 g/L, 7 g/L, 8 g/L, 9 g/L, 10 g/L, 12 g/L, 15g/L, 20 g/L, 25 g/L, or 30 g/L. In some embodiments, a sample containingthe antibody of interest may be loaded onto an HA resin to a density onthe resin of between at least 1 g/L, 2 g/L, 3 g/L, 4 g/L, 5 g/L, 6 g/L,7 g/L, 8 g/L, 9 g/L, 10 g/L, 12 g/L, 15 g/L, 20 g/L, or 25 g/L and nomore than 2 g/L, 3 g/L, 4 g/L, 5 g/L, 6 g/L, 7 g/L, 8 g/L, 9 g/L, 10g/L, 12 g/L, 15 g/L, 20 g/L, 25 g/L, or 30 g/L, wherein the second valueis larger than the first value.

Washing the HA Column

After loading the sample containing the antibody of interest andimpurities on to the HA column, but prior to elution of the antibody ofinterest, methods provided herein may optionally comprise an additionalstep of washing the loaded column with one or more wash buffer(s) to,for example, remove non-specifically immobilized impurities or otherwiseprepare or equilibrate the column for the elution step. The propertiesof any wash buffer can be determined by one of ordinary skill in theart. In one embodiment the wash buffer is a phosphate buffer comprising,for example, sodium phosphate, and the concentration of the phosphateions in the buffer is from about 5 to 50 mM. For example, in anembodiment, a wash buffer may contain about 10 to 40 mM phosphate ions;in other embodiments, it may contain about 10, 20, 30, 40, or 50 mMphosphate ions. Optionally, a wash buffer may additionally contain HEPESin a concentration from about 1 to 50 mM. For example, in an embodiment,a wash buffer may contain about 2 to 30 mM HEPES; in other embodiments,it may contain about 5, 10, 15, 20, or 25 mM HEPES. A wash buffer mayhave a pH of about 6.0 to 9.0. For example, in an embodiment, a washbuffer may have a pH of about 7.0 to 8.0; in other embodiments, it mayhave a pH of about 7.0, 7.5, or 8.0. In one embodiment, a wash buffercontains about 40 mM phosphate ions, 20 mM HEPES, and has a pH of about7.5.

Eluting the Antibody of Interest from the HA Column

The methods provided herein comprise the step of eluting the boundantibody of interest from the HA resin. The bound antibody of interestis eluted by one or more elution buffers. Typically, the elution buffercontains one or more salts or ions, and the concentration of the saltsor ions is increased during the elution.

In some embodiments, an elution buffer provided herein comprisesphosphate ions. Optionally, the concentration of phosphate ions in theelution buffer is increased from an initial concentration of about 20 mMto about 200 mM during the elution. For example, in an embodiment, theconcentration of phosphate ions in the elution buffer is increased froman initial concentration of about 40 mM to about 80 mM or about 40 mM toabout 100 mM during the elution. The specific manner and rate ofincreasing the concentration of phosphate ions in the elution buffer maybe determined as is suitable for the antibody of interest, and thattakes into account the types of impurity molecules that are also boundto the HA resin. For example, the concentration of phosphate ions in theelution buffer may be raised in a gradual/shallow linear gradient. Useof a shallow gradient may permit the effective separation of one or moremolecules that elute from the HA resin under similar, but differentconditions. Alternatively, in some embodiments, the concentration ofphosphate ions in the elution buffer may be raised in a steep gradient,or it may be raised stepwise. Optionally, an elution buffer mayadditionally contain HEPES in a concentration from about 1 to 50 mM. Forexample, in an embodiment, an elution buffer may contain about 2 to 30mM HEPES; in other embodiments, it may contain about 5, 10, 15, 20, or25 mM HEPES. An elution buffer may have a pH of about 6.0 to 9.0. Forexample, in an embodiment, an elution buffer may have a pH of about 7.0to 8.0; in other embodiments, it may have a pH of about 7.0, 7.5, or8.0. In one embodiment, an elution buffer contains about 40-80 mMphosphate ions (increasing over a gradient), 20 mM HEPES, and has a pHof about 7.5.

The elution conditions, including, but not limited to the properties ofthe elution buffer suitable for use with a HA resin (such as the buffercomposition, pH, concentration, ionic strength, and the like); anynecessary step or gradient change in the properties of the elutionbuffer; number of column volumes of elution buffer to be used; flow rateand the like can be determined to optimize the elution of the antibodyof interest from the HA column, as well as the separation of theantibody of interest from impurity molecules.

Following elution, the one or more peak fractions containing theantibody of interest are optionally collected individually or separatelyand optionally pooled, the pH optionally adjusted, optionally filteredand then optionally stored prior to additional processing as desired.The peak fractions for collection can be identified by any suitablemeans, such as identification using ultraviolet at A280 and startingcollection when the ultraviolet signal rises above a desired amountand/or at a desired point in the elution conditions.

Material eluted from the HA resin and containing the antibody ofinterest may be optionally be referred to herein as a “purifiedfraction” or the like. The purified fraction may contain material from asingle fraction eluted from the HA resin, or it may be the combinationof multiple fractions eluted from the HA resin that have been pooledtogether. Typically, the purified fraction is prepared such that it isbalanced between collecting a high amount of the antibody of interest,but a low amount of impurity molecules. These competing goals must oftenbe balanced, for example, because there may be an at least partialoverlap between the conditions when the antibody of interest elutes fromthe HA resin, and when a species of impurity molecule elutes from the HAresin.

Any of the buffers for methods provided herein (e.g. an equilibrationbuffer, load buffer, wash buffer, or elution buffer) may also compriseadditional or alternative suitable components such as acetate,succinate, MES, ACES, MOPSO, PIPES, BES, TAPSO, AMPSO, TRICINE, EPPS,Bicine, DIPSO, HEPPSO, imidazole, Tris, Bis-tris, TAPS, arginine,glycine, acetonitrile, ethanol, methanol, 1% sodium dodecyl sulfate(SDS) or other surfactants, and the like.

In some embodiments, any of the buffers provided herein may have a pH ofabout 6.0 to 9.0. In other embodiments, any of the buffers providedherein may have a pH of about 5.0 to 9.0, 5.5 to 9.0, 6.5 to 9.0, 7.0 to9.0, 7.5 to 9.0, 7.0 to 8.0, or 6.5 to 8.5.

In buffers provided herein described as containing “phosphate ions”, thephosphate ions may be generated in the buffer from any suitablephosphate salt, such as sodium phosphate or potassium phosphate. Inaddition, solutions provided herein that are described as being preparedwith “sodium phosphate” may be prepared with any suitable sodiumphosphate salt (e.g. monobasic or dibasic).

After the antibody of interest has been eluted from the HA column, theHA column is optionally cleaned to remove impurities and othercomponents which degrade the column resin and prepare it for storagesubsequent use. In one embodiment, the column is first regenerated usinga buffer such as one containing sodium phosphate at a concentration ofabout 0.4 M and at a pH of about 7.5; followed by an optionalsanitization step using a cleaning solution such as about 1 M NaOH andabout 0.5 M potassium phosphate, and then prepared for storage using astorage solution such as about 0.1 M NaOH.

Antibodies

Methods provided herein may be used to purify an antibody of interestfrom one or more impurities. For example, the purified antibody can beused as or in the preparation of pharmaceuticals.

In some embodiments, an antibody purified according to a method providedherein is any type of antibody provided herein. For example, an antibodypurified according to a method provided herein may be a full-lengthantibody or an antibody fragment (e.g. an scFv or Fab), and it may bemonospecific or bispecific. Typically, an antibody of interest purifiedaccording to a method provided herein is a recombinant antibody.

IgG Antibodies

In some embodiments, an antibody that may be purified according to amethod provided herein is an immunoglobulin G (IgG) antibody. As isknown in the art, an IgG antibody contains two heavy chains and twolight chains, and has a general “Y” shape. In standard IgG molecules,the two heavy chains have the same amino acid sequence, and the twolight chains have the same amino acid sequence. An IgG antibody may bedescribed as having two “arms” (i.e. a “first arm” and a “second arm”),in which each arm contains one heavy chain and one light chain, linkedtogether by a disulfide bond. In standard IgG molecules, the first armof the antibody is identical to the second arm of the antibody (due toeach arm containing a heavy chain and light chain that have the sameamino acid sequence as the heavy chain and light chain in the other arm,respectively). The N-terminal region of the heavy chain contains theheavy chain variable region (VH), and the N-terminal region of the lightchain contains the light chain variable region (VL). The VH and VLregions contain the portion of the antibody that specifically binds toan antigen. Thus, each arm of an IgG antibody can specifically bind toan antigen. In standard IgG molecules, both the first arm and the secondarm of the IgG antibody bind to the same antigen (due to the fact thatboth arms contain heavy chains and light chains having the samerespective amino acid sequence). A standard IgG antibody may be referredto as being “homodimeric”, based on having 2 arms that are the same. AnIgG antibody purified according to a method provided herein may be ofthe subclass IgG1, IgG2, IgG3, or IgG4.

Bispecific IgG Antibodies

In some embodiments, an antibody that may be purified according to amethod provided herein is a bispecific IgG antibody. In a bispecific IgGantibody, each of the two arms of the antibody specifically binds to adifferent antigen. In addition, the amino acid sequence of the heavychain in the first arm of the bispecific IgG antibody is different fromthe amino acid sequence of the heavy chain in the second arm of the samebispecific IgG antibody, and similarly, the amino acid sequence of thelight chain in the first arm of the bispecific IgG antibody is typicallydifferent from the amino acid sequence of the light chain in the secondarm of the same bispecific IgG antibody. A bispecific IgG antibody maytherefore be referred to as being “heterodimeric”, based on having 2arms that are different. The first arm of a bispecific IgG antibody maybe described as being specific for a “first antigen”, and the second armof a bispecific IgG antibody may be described as being specific for a“second antigen”. In some embodiments, the bispecific antibody has anIgG1, IgG2, IgG3, or IgG4 isotype. In some embodiments, the bispecificantibody comprises an immunologically inert Fc region.

Bispecific IgG Antibodies—Methods of Making

Methods for making bispecific antibodies are known in the art (see,e.g., Suresh et al., Methods in Enzymology 121:210, 1986).Traditionally, the recombinant production of bispecific antibodies wasbased on the coexpression of two immunoglobulin heavy chain-light chainpairs, with the two heavy chains having different specificities(Millstein and Cuello, Nature 305, 537-539, 1983).

More recently, methods have been developed in which the followinggeneral steps are taken to prepare bispecific heterodimeric antibodies:

1) A first homodimeric antibody (also referred to herein as a “firstparent antibody”) and a second homodimeric antibody (also referred toherein as a “second parent antibody”) are individually expressed andpurified. The first homodimeric antibody is specific for a first targetantigen of the bispecific antibody being prepared, and the secondhomodimeric antibody is specific for a second target antigen of thebispecific antibody being prepared. Thus, for example, if the objectiveis to prepare a bispecific antibody having specificity for BCMA and CD3,a monoclonal anti-BCMA antibody (the “first parent antibody”) and amonoclonal anti-CD3 antibody (the “second parent antibody”) areseparately expressed and purified.

2) Next, the purified first homodimeric/parent antibody and the purifiedsecond homodimeric/parent antibody are mixed and incubated togetherunder conditions that promote antibody arm exchange, such thatheterodimeric bispecific antibodies are formed that contain a first armfrom the first parent antibody and a second arm from the second parentantibody. These conditions typically involve a sequence of reducingconditions followed by oxidizing conditions. The reducing conditionspromote cleavage of the disulfide bonds holding the two heavy chains ofthe homodimeric antibodies together, and thereby permit switching ofantibody arms between the first parent antibody and second parentantibody. The subsequent oxidizing conditions then form new disulfidebridges which stabilize the newly-formed bispecific antibodies. Thisgeneral approach for generating bispecific antibodies is outlined inFIG. 1. In FIG. 1, a first parent antibody (“Parent Antibody A”; greycolor) and a second parent antibody (“Parent Antibody B”; black color)are depicted, each of which is a monospecific homodimer, and contains afirst arm and a second arm. Typically, the first parent antibody and thesecond parent antibody are specific for different antigens. Then, thefirst parent antibody and second parent antibody are mixed together andexposed to reduction and oxidation steps that result in the formation ofthe bispecific antibody of interest, which contains a first arm from theParent Antibody A, and a second arm from the Parent Antibody B, and therespective specificities of both arms.

Optionally, the amino acid sequence of an antibody heavy chain may bemodified in one or more ways to promote the formation of bispecificantibodies. For example, the heavy chain of one arm of a bispecificantibody may contain an amino acid modification in the first hingeregion, such that the substituted/replaced amino acid in the first hingeregion has an opposite charge to the corresponding amino acid in thehinge region of the other arm of the formed bispecific antibody. This isdescribed, for example, in International Patent Application No.PCT/US2011/036419 (WO2011/143545). In another approach, the formation ofa desired heteromultimeric or heterodimeric protein (e.g., bispecificantibody) is enhanced by altering or engineering an interface between afirst and a second immunoglobulin-like Fc region (e.g., a hinge regionand/or a CH3 region). In this approach, the bispecific antibody maycontain a CH3 region, wherein the CH3 region comprises a first CH3polypeptide and a second CH3 polypeptide which interact together to forma CH3 interface, wherein one or more amino acids within the CH3interface destabilize homodimer formation and are not electrostaticallyunfavorable to homodimer formation. This approach is also described inInternational Patent Application No. PCT/US2011/036419 (WO2011/143545).

The above method and other methods for preparing bispecific antibodiesare further described, for example, in: International Patent ApplicationNo. PCT/IB2011/054899 (WO2012/059882), PCT/US2011/036419(WO2011/143545), and Giese et al, Biotechnology Progress, “BispecificAntibody Process Development: Assembly and Purification of Knob and HoleBispecific Antibodies”, 17 Jan. 2018, and references cited therein, eachof which are incorporated by reference herein for all purposes. Methodsprovided herein for the purification of antibodies may be used to purifybispecific antibodies that were prepared by any suitable method.

Bispecific IgG Antibodies—Specificity

In some embodiments, an antibody that may be purified according to amethod provided herein is a full-length human bispecific IgG antibody,wherein a first antibody variable domain of the first arm of thebispecific antibody is capable of binding to a first antigen, and asecond antibody variable domain of the second arm of the bispecificantibody is capable of binding to a second antigen. The first antigenand second antigen may have any characteristics of an antigen asdescribed herein. In some embodiments, the first antigen occurs on afirst cell type, and the second antigen on a second cell type.

In some embodiments, an antibody that may be purified according to amethod provided herein is a full-length human bispecific IgG antibody,wherein a first antibody variable domain of the antibody is capable ofrecruiting the activity of a human immune effector cell by specificallybinding to an effector antigen located on the human immune effectorcell, and wherein a second antibody variable domain of the antibody iscapable of specifically binding to a target antigen.

A human immune effector cell that can be bound by an antibody providedherein can be any of a variety of immune effector cells known in theart. For example, the immune effector cell can be a member of the humanlymphoid cell lineage, including, but not limited to, a T cell (e.g., acytotoxic T cell), a B cell, and a natural killer (NK) cell. The immuneeffector cell can also be, for example, a member of the human myeloidlineage, including, but not limited to, a monocyte, a neutrophilicgranulocyte, and a dendritic cell. Such immune effector cells may haveeither a cytotoxic or an apoptotic effect on a target cell or otherdesired effect upon activation by binding of an effector antigen. Theeffector antigen is an antigen (e.g., a protein or a polypeptide) thatis expressed on the human immune effector cell. Examples of effectorantigens that can be bound by an antibody provided herein include, butare not limited to, human CD3 (or CD3 (Cluster of Differentiation)complex), CD16, NKG2D, NKp46, CD2, CD28, CD25, CD64, and CD89.

The target antigen is expressed on a target cell in a diseased condition(e.g., an inflammatory disease, a proliferative disease (e.g., cancer),an immunological disorder, a neurological disease, a neurodegenerativedisease, an autoimmune disease, an infectious disease (e.g., a viralinfection or a parasitic infection), an allergic reaction, agraft-versus-host disease or a host-versus-graft disease). A targetantigen is not effector antigen. Examples of the target antigensinclude, but are not limited to, BCMA, EpCAM (Epithelial Cell AdhesionMolecule), CCR5 (Chemokine Receptor type 5), CD19, HER (Human EpidermalGrowth Factor Receptor)-2/neu, HER-3, HER-4, EGFR (Epidermal GrowthFactor Receptor), FLT3 (Fms-Like Tyrosine kinase 3), PSMA, CEA, MUC-1(Mucin), MUC2, MUC3, MUC4, MUC5AC, MUC5B, MUC7, CIhCG, Lewis-Y, CD20,CD33, CD30, ganglioside GD3, 9-O-Acetyl-GD3, GM2, Globo H, fucosyl GM1,Poly SA, GD2, Carboanhydrase IX (MN/CA IX), CD44v6, Shh (SonicHedgehog), Wue-1, Plasma Cell Antigen, (membrane-bound) IgE, MCSP(Melanoma Chondroitin Sulfate Proteoglycan), CCR8, TNF-alpha precursor,STEAP, mesothelin, A33 Antigen, PSCA (Prostate Stem Cell Antigen), Ly-6;desmoglein 4, E-cadherin neoepitope, Fetal Acetylcholine Receptor, CD25,CA19-9 marker, CA-125 marker and MIS (Muellerian Inhibitory Substance)Receptor type II, sTn (sialylated Tn antigen; TAG-72), FAP (fibroblastactivation antigen), endosialin, EGFRvIII, LG, SAS and CD63.

In some embodiments, an antibody purified according to a method providedherein may be any antibody as described in U.S. application Ser. No.15/085,644, filed Mar. 30, 2016 (Publication No. US20160297885), or U.S.application Ser. No. 15/993,874, filed May 31, 2018 (Publication No.US20180346601), which are hereby incorporated by reference in theirentirety for all purposes.

In some embodiments, an antibody purified according to a method providedherein may be a bispecific IgG antibody, in which one arm of theantibody specifically binds to Cluster of Differentiation 3 (CD3).Information about CD3 is provided, for example, via UniProtKB ID#P07766.

In some embodiments, in a bispecific IgG antibody in which one arm ofthe antibody specifically binds to CD3, the VH region of the heavy chainof the CD3-binding arm has an amino acid sequence comprising the aminoacid sequence:

(SEQ ID NO: 1) EVQLVESGGGLVQPGGSLRLSCAASGFTFSDYYMTVVVRQAPGKGLEVVVAFIRNRARGYTSDHNPSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARDRPSYYVLDYWGQGTTVTVSS.In some embodiments, in a bispecific IgG antibody in which one arm ofthe antibody specifically binds to CD3, the heavy chain of theCD3-binding arm has an amino acid sequence comprising the amino acidsequence:

(SEQ ID NO: 2) EVQLVESGGGLVQPGGSLRLSCAASGFTFSDYYMTVVVRQAPGKGLEVVVAFIRNRARGYTSDHNPSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARDRPSYYVLDYWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCRVRCPRCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVAVSHEDPEVQFNVVYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPSSIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSRLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.In some embodiments, in a bispecific IgG antibody in which one arm ofthe antibody specifically binds to CD3, the VH region of the heavy chainof the CD3-binding arm has an amino acid sequence comprising a CDR1, aCDR2, and a CDR3 of the VH sequence shown in SEQ ID NO: 1.

In some embodiments, in a bispecific IgG antibody in which one arm ofthe antibody specifically binds to CD3, the VL region of the light chainof the CD3-binding arm has an amino acid sequence comprising the aminoacid sequence:

(SEQ ID NO: 3) DIVMTQSPDSLAVSLGERATINCKSSQSLFNVRSRKNYLAVVYQQKPGQPPKLLISWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAV YYCKQSYDLFTFGSGTKLEIK.In some embodiments, in a bispecific IgG antibody in which one arm ofthe antibody specifically binds to CD3, the light chain of theCD3-binding arm has an amino acid sequence comprising the amino acidsequence:

(SEQ ID NO: 4) DIVMTQSPDSLAVSLGERATINCKSSQSLFNVRSRKNYLAVVYQQKPGQPPKLLISWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCKQSYDLFTFGSGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC.In some embodiments, in a bispecific IgG antibody in which one arm ofthe antibody specifically binds to CD3, the VL region of the light chainof the CD3-binding arm has an amino acid sequence comprising a CDR1, aCDR2, and a CDR3 of the VL sequence shown in SEQ ID NO: 3.

In some embodiments, in a bispecific IgG antibody in which one arm ofthe antibody specifically binds to CD3, the VH region of the heavy chainof the CD3-binding arm has an amino acid sequence comprising the aminoacid sequence shown in SEQ ID NO: 1, and the VL region of the lightchain of the CD3-binding arm has an amino acid sequence comprising theamino acid sequence shown in SEQ ID NO: 3. In some embodiments, in abispecific IgG antibody in which one arm of the antibody specificallybinds to CD3, the heavy chain of the CD3-binding arm has an amino acidsequence comprising the amino acid sequence shown in SEQ ID NO: 2, andthe light chain of the CD3-binding arm has an amino acid sequencecomprising the amino acid sequence shown in SEQ ID NO: 4. In someembodiments, in a bispecific IgG antibody in which one arm of theantibody specifically binds to CD3, the VH region of the heavy chain ofthe CD3-binding arm has an amino acid sequence comprising a CDR1, aCDR2, and a CDR3 of the VH sequence shown in SEQ ID NO: 1, and the VLregion of the light chain of the CD3-binding arm has an amino acidsequence comprising a CDR1, a CDR2, and a CDR3 of the VL sequence shownin SEQ ID NO: 3.

In some embodiments, an antibody purified according to a method providedherein may be a bispecific IgG antibody, in which one arm of theantibody specifically binds to B-cell maturation antigen (BCMA).Information about BCMA is provided, for example, via UniProtKB ID#Q02223.

In some embodiments, in a bispecific IgG antibody in which one arm ofthe antibody specifically binds to BCMA, the VH region of the heavychain of the BCMA-binding arm has an amino acid sequence comprising theamino acid sequence:

(SEQ ID NO: 5) EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYPMSVVVRQAPGKGLEVVVSAIGGSGGSLPYADIVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARYWPMDIWGQGTLVTVSS.In some embodiments, in a bispecific IgG antibody in which one arm ofthe antibody specifically binds to BCMA, the heavy chain of theBCMA-binding arm has an amino acid sequence comprising the amino acidsequence:

(SEQ ID NO: 6) EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYPMSVVVRQAPGKGLEVVVSAIGGSGGSLPYADIVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARYWPMDIWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCEVECPECPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVAVSHEDPEVQFNVVYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPSSIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCEVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.In some embodiments, in a bispecific IgG antibody in which one arm ofthe antibody specifically binds to BCMA, the VH region of the heavychain of the BCMA-binding arm has an amino acid sequence comprising aCDR1, a CDR2, and a CDR3 of the VH sequence shown in SEQ ID NO: 5.

In some embodiments, in a bispecific IgG antibody in which one arm ofthe antibody specifically binds to BCMA, the VL region of the lightchain of the BCMA-binding arm has an amino acid sequence comprising theamino acid sequence:

(SEQ ID NO: 7) EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAVVYQQKPGQAPRLLMYDASIRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYC QQYQSWPLTFGQGTKVEIK.In some embodiments, in a bispecific IgG antibody in which one arm ofthe antibody specifically binds to BCMA, the light chain of theBCMA-binding arm has an amino acid sequence comprising the amino acidsequence:

(SEQ ID NO: 8) EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAVVYQQKPGQAPRLLMYDASIRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYQSWPLTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC.In some embodiments, in a bispecific IgG antibody in which one arm ofthe antibody specifically binds to BCMA, the VL region of the lightchain of the BCMA-binding arm has an amino acid sequence comprising aCDR1, a CDR2, and a CDR3 of the VL sequence shown in SEQ ID NO: 7.

In some embodiments, in a bispecific IgG antibody in which one arm ofthe antibody specifically binds to BCMA, the VH region of the heavychain of the BCMA-binding arm has an amino acid sequence comprising theamino acid sequence shown in SEQ ID NO: 5, and the VL region of thelight chain of the BCMA-binding arm has an amino acid sequencecomprising the amino acid sequence shown in SEQ ID NO: 7. In someembodiments, in a bispecific IgG antibody in which one arm of theantibody specifically binds to BCMA, the heavy chain of the BCMA-bindingarm has an amino acid sequence comprising the amino acid sequence shownin SEQ ID NO: 6, and the light chain of the BCMA-binding arm has anamino acid sequence comprising the amino acid sequence shown in SEQ IDNO: 8. In some embodiments, in a bispecific IgG antibody in which onearm of the antibody specifically binds to BCMA, the VH region of theheavy chain of the BCMA-binding arm has an amino acid sequencecomprising a CDR1, a CDR2, and a CDR3 of the VH sequence shown in SEQ IDNO: 5, and the VL region of the light chain of the BCMA-binding arm hasan amino acid sequence comprising a CDR1, a CDR2, and a CDR3 of the VLsequence shown in SEQ ID NO: 7.

In some embodiments, in a bispecific IgG antibody in which one arm ofthe antibody specifically binds to BCMA, the VH region of the heavychain of the BCMA-binding arm has an amino acid sequence comprising theamino acid sequence:

(SEQ ID NO: 13) EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYPMSVVVRQAPGKGLEVVVSAIGGSGGSLPYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARYWPMDIWGQGTLVTVSS.In some embodiments, in a bispecific IgG antibody in which one arm ofthe antibody specifically binds to BCMA, the VL region of the lightchain of the BCMA-binding arm has an amino acid sequence comprising theamino acid sequence:

(SEQ ID NO: 14) EIVLTQSPGTLSLSPGERATLSCRASQSVSSTYLAVVYQQKPGQAPRLLMYDASIRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQ YQEWPLTFGQGTKVEIK.In some embodiments, in any reference herein to an antibody comprisingthe a VH region that has an amino acid sequence comprising the aminoacid sequence shown in SEQ ID NO: 5, the antibody may alternativelycomprise a VH region comprising the amino acid sequence shown in SEQ IDNO: 13. In some embodiments, in any reference herein to an antibodycomprising the a VL region that has an amino acid sequence comprisingthe amino acid sequence shown in SEQ ID NO: 7, the antibody mayalternatively comprise a VL region comprising the amino acid sequenceshown in SEQ ID NO: 14. Similarly, also included herein are anti-BCMAheavy and light chains containing the VH and VL sequence of SEQ ID NO:13 and SEQ ID NO: 14, respectively.

In some embodiments, an antibody purified according to a method providedherein may be a bispecific IgG antibody, in which one arm of theantibody specifically binds to fms-like tyrosine kinase 3 (FLT3).Information about FLT3 is provided, for example, via UniProtKB ID#P36888.

In some embodiments, in a bispecific IgG antibody in which one arm ofthe antibody specifically binds to FLT3, the VH region of the heavychain of the FLT3-binding arm has an amino acid sequence comprising theamino acid sequence:

(SEQ ID NO: 9) EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMNVVVRQAPGKGLEVVVSAISGGGRSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDLSPSDVGWGYGFDIWGQGTLVTVSS.In some embodiments, in a bispecific IgG antibody in which one arm ofthe antibody specifically binds to FLT3, the heavy chain of theFLT3-binding arm has an amino acid sequence comprising the amino acidsequence:

(SEQ ID NO: 10) EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMNVVVRQAPGKGLEVVVSAISGGGRSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDLSPSDVGWGYGFDIWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCEVECPECPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVAVSHEDPEVQFNVVYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPSSIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCEVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG.In some embodiments, in a bispecific IgG antibody in which one arm ofthe antibody specifically binds to FLT3, the VH region of the heavychain of the FLT3-binding arm has an amino acid sequence comprising aCDR1, a CDR2, and a CDR3 of the VH sequence shown in SEQ ID NO: 9.

In some embodiments, in a bispecific IgG antibody in which one arm ofthe antibody specifically binds to FLT3, the VL region of the lightchain of the FLT3-binding arm has an amino acid sequence comprising theamino acid sequence:

(SEQ ID NO: 11) EIVLTQSPATLSLSPGERATLSCRASQSVSSNLAVVYQQKPGQAPRLLIYDTFTRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQYGS SPPTFGQGTRLEIK.In some embodiments, in a bispecific IgG antibody in which one arm ofthe antibody specifically binds to FLT3, the light chain of theFLT3-binding arm has an amino acid sequence comprising the amino acidsequence:

(SEQ ID NO: 12) EIVLTQSPATLSLSPGERATLSCRASQSVSSNLAVVYQQKPGQAPRLLIYDTFTRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQYGSSPPTFGQGTRLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC.In some embodiments, in a bispecific IgG antibody in which one arm ofthe antibody specifically binds to FLT3, the VL region of the lightchain of the FLT3-binding arm has an amino acid sequence comprising aCDR1, a CDR2, and a CDR3 of the VL sequence shown in SEQ ID NO: 11.

In some embodiments, in a bispecific IgG antibody in which one arm ofthe antibody specifically binds to FLT3, the VH region of the heavychain of the FLT3-binding arm has an amino acid sequence comprising theamino acid sequence shown in SEQ ID NO: 9, and the VL region of thelight chain of the FLT3-binding arm has an amino acid sequencecomprising the amino acid sequence shown in SEQ ID NO: 11. In someembodiments, in a bispecific IgG antibody in which one arm of theantibody specifically binds to FLT3, the heavy chain of the FLT3-bindingarm has an amino acid sequence comprising the amino acid sequence shownin SEQ ID NO: 10, and the light chain of the FLT3-binding arm has anamino acid sequence comprising the amino acid sequence shown in SEQ IDNO: 12. In some embodiments, in a bispecific IgG antibody in which onearm of the antibody specifically binds to FLT3, the VH region of theheavy chain of the FLT3-binding arm has an amino acid sequencecomprising a CDR1, a CDR2, and a CDR3 of the VH sequence shown in SEQ IDNO: 9, and the VL region of the light chain of the FLT3-binding arm hasan amino acid sequence comprising a CDR1, a CDR2, and a CDR3 of the VLsequence shown in SEQ ID NO: 11.

In some embodiments, provided herein is a bispecific anti-BCMA/anti-CD3antibody, in which the anti-BCMA arm of the antibody has any of thecharacteristics described above for an anti-BCMA arm, and the anti-CD3arm of the antibody has any of the characteristics described above foran anti-CD3 arm. In some embodiments, provided herein is a bispecificanti-FLT3/anti-CD3 antibody, in which the anti-FLT3 arm of the antibodyhas any of the characteristics described above for an anti-FLT3 arm, andthe anti-CD3 arm of the antibody has any of the characteristicsdescribed above for an anti-CD3 arm.

Also provided herein are methods of purifying a monospecific antibodyhaving affinity for any of the above antigens, and/or which contain anyof the amino acid sequences described above. For example, also providedherein is purification of a monospecific, homodimeric anti-CD3 antibodycomprising the VH amino acid sequence as shown in SEQ ID NO: 1.

Impurities

Methods provided herein may be used to purify an antibody of interestfrom one or more impurities.

Impurities include, for example, clipped versions of the antibody ofinterest, protein aggregates, and in the case of a bispecific antibodyof interest, parental monospecific antibodies related to the formationof the bispecific antibody of interest. These different impurities mayalso be referred to herein as different “species of impurity”, “impuritymolecules”, or the like.

Clipped Versions of an Antibody of Interest

“Clipped versions of an antibody of interest”, “clipped antibodies”, orthe like refer to a recombinant antibody in which one or morepolypeptide bonds in the antibody has been cleaved, as compared to acorresponding intact antibody of interest. In contrast, an “intact”antibody refers to a recombinant antibody that contains all of theexpected peptide bonds and amino acids of the recombinant antibody (i.e.that would be expected based on the nucleic acid sequence(s) encodingthe polypeptide(s) of the antibody)

As such, clipped antibodies may be considered to be degradation productsrelated to the antibody of interest. Cleavage of a peptide bond in anantibody may occur, for example, via enzymatic (e.g. protease-mediated)or non-enzymatic activities.

In some embodiments, when a peptide bond in a polypeptide of an antibodyis cleaved, after the cleavage, a cleaved portion of the polypeptidechain might no longer be covalently linked to the rest of the antibody;in that case, the cleaved portion of the polypeptide chain maydissociate from the rest of the antibody. This most commonly occurs whenthe cleavage is in a peptide bond near an N or C terminus of the apolypeptide chain, and it results in a clipped antibody which has lostone or more amino acids as compared to the corresponding intactantibody. These clipped antibodies have less mass than the correspondingintact antibody, due to the loss of one or more amino acids from theantibody.

Alternatively, in some other embodiments, when a peptide bond in apolypeptide of an antibody is cleaved, after the cleavage, a cleavedportion of the polypeptide chain might still remain covalently linked tothe rest of the antibody (for example, by an intra-chain or inter-chaindisulfide bond). In this case, even though there is a cleavage a peptidebond of the antibody, the cleaved portion of the polypeptide chain willremain tethered to the rest of the antibody, via the remaining intactcovalent bond(s) that link the cleaved portion of the polypeptide chainto the rest of the antibody. In this circumstance, the clipped antibodywill still have the same number of amino acids and amino acid sequencesas compared to the intact antibody. In addition, in at least someembodiments, this type of clipped antibody may have a slightly greatermass than the corresponding intact antibody. This gain of mass may bethe result, for example, of one or more chemical reactions that occurupon the cleavage of the peptide bond. During such reactions, one ormore atoms (e.g. H, O) may react with atoms of the antibody polypeptidechain and become covalently linked to the antibody chain, which resultsin a gain of mass by the clipped antibody as compared to thecorresponding intact antibody.

Since a “clipped” antibody is generated from a corresponding “intact”antibody, the “clipped” version of the antibody has the same amino acidsequence (in the case of no loss of amino acids from the antibody as theresult of the peptide bond cleavage) or nearly the same amino acidsequence (in the case of the loss of one or more amino acid sequencesfrom the antibody as the result of the peptide bond cleavage) as thecorresponding “intact” version of the antibody.

Typically, a clipped version of an antibody has a mass that is similarto the mass of the corresponding intact antibody. As described above, insome embodiments, a clipped antibody may have a mass that is less thanthe corresponding intact antibody (for example, in the event that theclipping results in the loss of one or more amino acids from theantibody). Alternatively, in some embodiments, a clipped antibody mayhave a mass that is greater than the corresponding intact antibody (inthe event that the clipping does not result in the loss of any aminoacids from the antibody, and instead, results in the gain of at least anatom by the antibody via one or more reactions that occur as a result ofthe cleavage of the peptide bond).

In some embodiments, a clipped version of an antibody has a mass that isno more than 50%, 40%, 30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, 1%,0.5%, 0.4%, 0.3%, 0.2%, 0.1%, 0.05%, 0.04%, 0.03%, 0.02%, 0.01%different than the mass of the corresponding intact antibody ofinterest. Put another way, in some embodiments, a clipped version of anantibody of interest has a mass that differs from the mass of thecorresponding intact antibody of interest by no more than 50%, 40%, 30%,25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%,0.05%, 0.04%, 0.03%, 0.02%, or 0.01%.

As described above, in some embodiments, a clipped version of anantibody of interest has a mass that is less than the correspondingintact antibody of interest. For example, in some embodiments, a clippedversion of an antibody has a mass that is no more than 50%, 40%, 30%,25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%,0.05%, 0.04%, 0.03%, 0.02%, 0.01% less than the mass of thecorresponding intact antibody of interest. In other words, if a clippedversion of an antibody has a mass that is no more than 10% less than themass of the corresponding intact antibody of interest, then, forexample, if the intact antibody of interest has a mass of 100,000 Da,then the clipped version of the antibody has a mass that is no more than10,000 Da less than that (10% of 100,000 is 10,000)—i.e. it has a massbetween 90,000 and 100,000 Da.

As also described above, in some embodiments, a clipped version of anantibody of interest has a mass that is greater than the correspondingintact antibody of interest. For example, in some embodiments, a clippedversion of an antibody has a mass that is no more than 5%, 4%, 3%, 2%,1%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, 0.05%, 0.04%, 0.03%, 0.02%, or 0.01%greater than the mass of the corresponding intact antibody of interest.In other words, if a clipped version of an antibody has a mass that isno more than 1% greater than the mass of the corresponding intactantibody of interest, then, for example, if the intact antibody ofinterest has a mass of 100,000 Da, then the clipped version of theantibody has a mass that is no more than 1,000 Da more than that (1% of100,000 is 1,000)—i.e. it has a mass between 100,000 and 101,000 Da.

High Molecular Mass Species (HMMS)/Protein Aggregates

In some embodiments, an impurity relevant to a method provided herein isreferred to as “high molecular mass species” (HMMS). HMMS refers to anyhigh molecular mass contaminant or impurity, but typically is anassociation of at least two proteins forming an aggregate. By way ofexample, HMMS may include multiple molecules of an antibody of interestthat have aggregated together and/or aggregates of proteins from hostcells that were used to produce an antibody of interest. Aggregates mayarise by any process including, for example, covalent or non-covalentlinking of molecules.

Parental Antibodies

In some embodiments, an impurity relevant to a method provided herein isa “parent antibody”, or “parental antibody” or the like. This type ofimpurity molecule is relevant to methods provided herein in which theantibody of interest is bispecific antibody that is generated from twodifferent parent antibodies, for example, as outlined in FIG. 1. Theseparent antibodies are monospecific, homodimers. Parent antibodies may bepresent in an antibody preparation with a bispecific antibody ofinterest due to multiple possible mechanisms, such as: i) in somesituations, some parent antibody molecules do not separate into a firstarm and a second arm during the reduction step to separate parentantibodies into a separate first arm and second arm (and thus, theantibodies remain as monospecific homodimers); or ii) in somesituations, a separated first arm and a second arm from the same type ofparent antibody join together, such they form a monospecific homodimer(rather than being involved in forming a heterodimer bispecificantibody). As used herein “parent antibody” refers to homodimericmolecules which occur by any of the above mechanisms. In addition, anantibody preparation as provided herein may contain as an impurity aparent antibody from one or both parent species.

Purification of an Antibody of Interest from Impurities

Provided herein are methods for purifying an antibody of interest fromone or more impurities.

In some embodiments, in a method provided herein, an antibody ofinterest is in an antibody preparation (also referred to herein as a“starting sample”) that contains the antibody of interest, as well asone or more species of impurity molecule. In this starting material fora method as provided herein, the antibody of interest may comprise, forexample, at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%,85%, 90%, or 95% by mass of the protein in the antibody preparation.Then, in some embodiments of a method provided herein, a purifiedfraction (also referred to herein as a “purified sample”) is collectedas eluate from the HA resin. This purified fraction contains theantibody of interest, and in some embodiments, still contains one ormore impurity molecules. In some embodiments, in a purified fractionprovided herein, the antibody of interest may comprise, for example, atleast about 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%,97%, 98%, or 99% by mass of the protein in the purified fraction.

In some embodiments, in a method provided herein, a starting sample isat least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, or95% by mass antibody of interest, and a subsequent purified sample inthe same method is at least about 20%, 30%, 40% 50% 60% 70% 75% 80% 85%90% 95% 96% 97%, 98%, or 99% by mass antibody of interest, wherein thesecond value is larger than the first value.

In some embodiments, in a method provided herein, a starting samplecontains at least about 0.1%, 0.5%, 1% 2% 3%, 4% 5%, 6%, 7% 8% 9%, 10%,15%, 20%, or 25% by mass clipped version of an antibody of interest. Insome embodiments, in a method provided herein, a purified samplecontains no more than about 0.01%, 0.05%, 0.1%, 0.5%, 1% 2%, 3%, 4%, 5%,6%, 7%, 8%, 9%, 10%, 15% or 20% by mass clipped version of an antibodyof interest. In some embodiments, in a method provided herein, astarting sample contains at least about 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%,6%, 7%, 8%, 9%, 10%, 15%, 20%, or 25% by mass clipped version of anantibody of interest, and a subsequent purified sample in the samemethod contains no more than about 0.01%, 0.05%, 0.1%, 0.5%, 1%, 2%, 3%,4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, or 20% by mass clipped version of anantibody of interest, wherein the second value is smaller than the firstvalue.

In some embodiments, in a method provided herein, a starting samplecontains an intact antibody of interest and a clipped version of theantibody of interest, wherein the ratio of clipped antibody to intactantibody is at least about 1:100, 1:50, 1:25, 1:20, 1:10, 1:5, 1:4, or1:3. In some embodiments, in a method provided herein, a purified samplecontains an intact antibody of interest and a clipped version of theantibody of interest, wherein the ratio of clipped antibody to intactantibody is no more than about 1:100, 1:50, 1:25, 1:20, or 1:10. In someembodiments, in a method provided herein, a starting sample contains anintact antibody of interest and a clipped version of the antibody ofinterest, wherein the ratio of clipped antibody to intact antibody inthe starting sample is at least about 1:100, 1:50, 1:25, 1:20, 1:10,1:5, 1:4, or 1:3 and wherein the ratio of clipped antibody to intactantibody in a subsequent purified sample in the same method is no morethan about 1:200, 1:100, 1:50, 1:25, 1:20, or 1:10, wherein the secondratio is smaller than the first ratio. In some embodiments, in a methodprovided herein, a starting sample contains an intact antibody ofinterest and a clipped version of the antibody of interest, wherein theratio of clipped antibody to intact antibody in the starting sample isbetween about one of the ratios in group A (group A ratios: 1:100, 1:50,1:25, 1:20, 1:10, 1:5, or 1:4) and one of the ratios in group B (group Bratios: 1:50, 1:25, 1:20, 1:10, 1:5, 1:4 or 1:3) and wherein the ratioof clipped antibody to intact antibody in a subsequent purified samplein the same method is no more than about 1:200, 1:100, 1:50, 1:25, 1:20,or 1:10, wherein the ratio in the purified sample is smaller than in thestarting sample.

In some embodiments, a starting sample provided according to a methodprovided herein contains at least 1, 5, 10, 15, 20, 25, 50, 100, 200,500, 1000, 2000, 5000, or 10,000 grams of the intact antibody ofinterest. In some embodiments, a purified sample provided according to amethod provided herein contains at least 1, 5, 10, 15, 20, 25, 50, 100,200, 500, 1000, 2000, 5000, or 10,000 grams of the intact antibody ofinterest. In some embodiments, a starting sample provided according to amethod provided herein contains at least 5, 10, 15, 20, 25, 50, 100,200, 500, 1000, 2000, 5000, or 10,000 grams of the intact antibody ofinterest, and a subsequent purified sample in the same method containsat least 1, 5, 10, 15, 20, 25, 50, 100, 200, 500, 1000, 2000, or 5000grams of the intact antibody of interest, wherein the first value islarger than the second value.

In addition, any above descriptions relating to the a) the amount or b)purity of an antibody of interest in a starting sample or purifiedsample may be taken together in reference to the same sample. Forexample, as stated above, a starting sample may contain at least about80% by mass antibody of interest; in addition, as also stated above, astarting sample may contain at least about 10 grams antibody ofinterest. Accordingly, also provided herein is a starting sample thatcontains at least about 80% by mass antibody of interest, and at least10 grams of antibody of interest, etc.

General Techniques

The practice of the present invention will employ, unless otherwiseindicated, conventional techniques of molecular biology (includingrecombinant techniques), microbiology, cell biology, biochemistry andimmunology, which are within the skill of the art. Such techniques areexplained fully in the literature, such as, Molecular Cloning: ALaboratory Manual, second edition (Sambrook et al., 1989) Cold SpringHarbor Press; Oligonucleotide Synthesis (M. J. Gait, ed., 1984); Methodsin Molecular Biology, Humana Press; Cell Biology: A Laboratory Notebook(J. E. Cellis, ed., 1998) Academic Press; Animal Cell Culture (R. I.Freshney, ed., 1987); Introduction to Cell and Tissue Culture (J. P.Mather and P. E. Roberts, 1998) Plenum Press; Cell and Tissue Culture:Laboratory Procedures (A. Doyle, J. B. Griffiths, and D. G. Newell,eds., 1993-1998) J. Wiley and Sons; Methods in Enzymology (AcademicPress, Inc.); Handbook of Experimental Immunology (D. M. Weir and C. C.Blackwell, eds.); Gene Transfer Vectors for Mammalian Cells (J. M.Miller and M. P. Calos, eds., 1987); Current Protocols in MolecularBiology (F. M. Ausubel et al., eds., 1987); PCR: The Polymerase ChainReaction, (Mullis et al., eds., 1994); Current Protocols in Immunology(J. E. Coligan et al., eds., 1991); Short Protocols in Molecular Biology(Wiley and Sons, 1999); Immunobiology (C. A. Janeway and P. Travers,1997); Antibodies (P. Finch, 1997); Antibodies: a practical approach (D.Catty., ed., IRL Press, 1988-1989); Monoclonal antibodies: a practicalapproach (P. Shepherd and C. Dean, eds., Oxford University Press, 2000);Using antibodies: a laboratory manual (E. Harlow and D. Lane (ColdSpring Harbor Laboratory Press, 1999); The Antibodies (M. Zanetti and J.D. Capra, eds., Harwood Academic Publishers, 1995), as well as insubsequent editions and corresponding websites of the above references,as applicable.

The following examples are offered for illustrative purposes only, andare not intended to limit the scope of the present invention in any way.Indeed, various modifications of the invention in addition to thoseshown and described herein will become apparent to those skilled in theart from the foregoing description and fall within the scope of theappended claims.

EXAMPLES Example 1: Purification of an Anti-BCMA/Anti-CD3 BispecificAntibody by cHA Resin Chromatography Objective:

In this example, methods for separating a full-length bispecific humanIgG of interest from various impurities were examined. The antibody ofinterest was a heterodimeric bispecific anti-BCMA/anti-CD3 antibody(i.e. one arm of the bispecific antibody was specific for BCMA, and theother arm was specific for CD3). The impurities present with thebispecific antibody of interest at the start of the purificationincluded: i) a clipped version of the intact anti-BCMA/anti-CD3bispecific antibody of interest; ii) homodimeric, monospecific anti-BCMAparent antibodies; iii) homodimeric, monospecific anti-CD3 antibodies;and iv) protein aggregates/high molecular mass species (HMMS).

Purification of the intact bispecific antibody of interest from theclipped version of the bispecific antibody presented a particularchallenge, as the clipped version of the bispecific antibody containedthe same number of amino acids and same amino acid sequences as theintact bispecific antibody of interest, and differed from the mass ofthe intact bispecific antibody by only 18 Daltons (Da). Specifically,the mass of the intact anti-BCMA/CD3 bispecific antibody is 148095.5 Da,while the mass of the corresponding clipped version of the bispecificantibody is 148113.5 (determined by mass spectrometry). Thus, theclipped bispecific antibody has a difference in mass of less than 0.1%(even less than 0.02%) as compared to the mass of the intact bispecificantibody (i.e. 0.1% of 148095 Da is 148 Da; 0.02% of 148095 Da is 29.6Da). Put another way, the mass of the clipped bispecific antibody isabout 100.01% of the mass of the intact bispecific antibody of interest.Accordingly, the mass of the clipped bispecific antibody is very similarto that of the intact bispecific antibody of interest.

The clipped version of the bispecific antibody contains a cleavage in apeptide bond of the anti-CD3 heavy chain, between the 56^(th) and57^(th) amino acid in the heavy chain. The anti-CD3 heavy chain has anamino acid sequence as shown in SEQ ID NO: 2, and accordingly, thecleavage occurs between the amino acids R and G in the “RG” sequence ofSEQ ID NO: 2 (“RG” only occurs once in SEQ ID NO: 2). It is believedthat this cleavage results in the gain of 1 oxygen atom and 2 hydrogenatoms in the clipped bispecific antibody (which would correspond to thegain of mass of 18 Da), as compared to the intact bispecific antibody.Also, although there is a cleaved peptide bond in the anti-CD3 heavychain, the cleaved 56-amino acid portion of the heavy chain (i.e. thefirst 56 amino acids of the chain) remains bound to the rest of theantibody via an intact, remaining intra-chain disulfide bond. Theintra-chain disulfide bond is between C22 and C98 of the anti-CD3 heavychain (i.e. C22 and C98 of the sequence shown in SEQ ID NO: 2). Thecontinued tethering of the cleaved 56-amino acid portion to the rest ofthe antibody by the intra-chain peptide bond is shown schematically inFIG. 2.

In FIG. 2, the schematic on the left depicts an intact bispecificantibody of interest, which contains intact heavy and light chains forboth the anti-CD3 and anti-BCMA arms of the bispecific antibody (in thefigure, the longer chains represent the heavy chains of the antibody,and the shorter chains represent the light chains). In addition, FIG. 2also shows various intra-antibody disulfide bonds, including intra-chaindisulfide bonds (e.g. linking different amino acids in the same lightchain or heavy chain), and well as inter-chain disulfide bonds (e.g.linking the heavy chain of the anti-BCMA arm to the heavy chain of theanti-CD3 arm, or linking the light chain of the anti-CD3 arm to theheavy chain of the anti-CD3 arm).

Another challenge presented in developing a method of purifying theintact bispecific antibody of interest from the various impurities wasthe objective of developing a method that would effectively separate thebispecific antibody of interest from impurities when relatively largeamounts of bispecific antibody of interest were to be purified. Forexample, one objective of the work related to this Example was todevelop a method for purification of a bispecific antibody that would beeffective for methods in which at least 1 gram of bispecific antibodywas to be purified. As is known in the art, purification of proteins ona large scale frequently presents numerous difficulties that are notpresent (or are significantly less of a problem) during purification ofthe same proteins on a small scale, due to, for example, difficulties inobtaining sharp chromatographic resolution between different proteinswhen performing chromatography on a large scale.

Materials and Methods:

The starting material for this work was an antibody preparationcontaining the bispecific IgG anti-BCMA/CD3 antibody of interest, aswell as various impurities, such as those noted above. The amino acidsequences of the polypeptides of the bispecific antibody are shown inthe following SEQ ID NOs: BCMA heavy chain: SEQ ID NO: 6; BCMA lightchain: SEQ ID NO: 8; CD3 heavy chain: SEQ ID NO: 2; CD3 light chain: SEQID NO: 4. The bispecific antibody of interest had been prepared from twoseparate parent antibodies, as described previously herein. Morespecifically, the parent monospecific anti-CD3 and anti-BCMA antibodieshad been purified via Protein-A chromatography, and these purifiedparent monospecific anti-CD3 and anti-BCMA antibodies had been used togenerate the bispecific anti-BCMA/CD3 antibody of interest. Thegenerated bispecific anti-BCMA/CD3 antibody had then been purified viaion exchange chromatography. The antibody preparation used as thestarting material for the purification work in this Example was eluatefrom the ion exchange column, which contained the bispecific antibody ofinterest and various remaining impurities, and it had buffers/salts atan approximate concentration of: 50 mM Tris and 60 mM glycine, pH 7.5.The antibody preparation contained over 85% by mass intact bispecificantibody of interest; it also contained about 8% clipped bispecificantibody, about 1% monospecific anti-BCMA parent antibodies, about 1monospecific anti-CD3 parent antibodies, and about 2% proteinaggregates/high molecular mass species (HMMS). Thus, while the antibodypreparation used as the starting material in this method containedintact bispecific antibody of interest that was already relatively pure,an objective of this method was to develop a method to increase thepurity of the intact bispecific antibody.

Results:

Multiple different chromatography resins and conditions were tested, inorder to try to identify a suitable resin and buffer conditions thatwould permit the effective purification of the intact anti-BCMA/CD3bispecific antibody of interest from the clipped bispecific antibody,the monospecific parent antibodies, and protein aggregates. During thisprocess, for example, multiple different ion-exchange and hydrophobicinteraction resins were tested, as well as various buffer and pHconditions. After extensive testing, hydroxyapatite resin was the onlyidentified resin that could permit effective purification of the intactanti-BCMA/CD3 bispecific antibody from various impurities, including theclipped bispecific antibody.

FIG. 3 shows a chromatographic profile of the elution of the bispecificantibody of interest from a ceramic hydroxyapatite (“cHA”) resin column,and the concurrent separation of the anti-BCMA/CD3 bispecific antibodyof interest from multiple different impurities, including the clippedversion of the bispecific antibody, both parent antibody species, andhigh molecular mass protein species. For the chromatography run depictedin FIG. 3, the antibody preparation that was loaded onto the cHA resinwas spiked with extra parent anti-BCMA and parent anti-CD3 antibodies,in order to more clearly identify the position of the elution of thesemolecules from the cHA column (however, no extra intact or clippedbispecific antibodies were added). In FIG. 3, the X-axis depicts, fromleft to right, the sequence of eluate from the cHA column (i.e. thematerial to the left is eluted from the cHA column earlier/at lower saltthan the material to the right). Typically, the eluate is collected insequential fractions from the column; thus, the X-axis may also beconsidered to depict the sequence of eluate fractions from the cHA resincolumn. The Y-axis depicts both UV absorbance (at 280 nM) andconductivity, as separately noted in the graph. The UV absorbancecorresponds to the presence of eluted protein, and the conductivitycorresponds to the salt concentration in the eluted material. Thus, FIG.3 depicts the profile of the elution of different proteins from the cHAresin column, as the salt concentration in the elution buffer flowingthrough the cHA resin increases. Following the UV graph in FIG. 3 fromleft to right, the graph shows various peaks and shoulders, whichcorrespond to either the intact bispecific anti-BCMA/CD3 antibody ofinterest, or various impurities. Specifically, from left to right, thefirst UV peak corresponds to the elution of the first parent antibody(“parent 1”; the monospecific anti-BCMA homodimer). The early portion ofthe next major peak (and the largest peak in the graph) corresponds tothe intact bispecific antibody protein of interest (“POI”). Thelater/tail end of that same major peak corresponds to the clippedversion of the bispecific antibody (“Clip”). While there is some overlapbetween the elution profile of the intact bispecific antibody ascompared to the clipped bispecific antibody, these two antibody typeselute from the cHA column under sufficiently different salt conditionsand fractions in order to significantly separate the intact bispecificantibody from the clipped version of the bispecific antibody. Finally,after the clipped version of the bispecific antibody elutes, the lastmajor peak/shoulder from the cHA column corresponds to the elution ofthe second parent antibody (monospecific anti-CD3 homodimer), as well ashigh molecular mass species (“HMMS”)(also referred to as proteinaggregates) from the cHA column. Thus, the graph of FIG. 3 shows thatthe intact anti-BCMA/CD3 antibody can be effectively purified frommultiple impurities including related clipped and parental antibodyspecies by cHA resin chromatography.

FIG. 4 provides a graph showing more detailed information about thedifferent molecular species present in the sequentially eluted fractionsfrom the cHA resin column, according to the cHA elution profile depictedin FIG. 3. Specifically, FIG. 4 provides detailed information about therelative amounts of: i) the intact anti-BCMA/CD3 bispecific antibodyprotein of interest (“POI”); ii) the clipped version of the bispecificantibody; iii) the first parent antibody/the monospecific anti-BCMAantibody; iv) the second parent antibody/the monospecific anti-CD3antibody; and v) high molecular mass species (“HMMS”) in the differentfractions eluted from the cHA column. The X-axis of FIG. 4 depicts, fromleft to right, the sequence of eluate from the cHA column (i.e. from lowsalt to high salt; each data point indicates a fraction eluted from thecolumn). The Y-axis of FIG. 4 depicts, on the left side, the percentageof each of i) the intact anti-BCMA/CD3 bispecific antibody protein ofinterest (“POI”); ii) the clipped version of the bispecific antibody;iii) the first parent antibody/the monospecific anti-BCMA antibody; andiv) the second parent antibody/the monospecific anti-CD3 antibody ineach fraction. The Y-axis of FIG. 4 depicts, on the right side, the %HMMS in each fraction.

The method as used to generate the data in FIG. 3 was also used with anantibody preparation that had not been spiked with any additionalantibodies; data from this chromatography run is presented in FIG. 5.Thus, the data in FIG. 5 reflects the purification via cHA resin of atypical antibody preparation eluted from an ion exchange column duringthe preparation of anti-BCMA/CD3 bispecific antibodies, which containsprimarily intact anti-BCMA/CD3 bispecific antibody of interest, as wellas various impurities, including the clipped version of the bispecificantibody. In FIG. 5, the X-axis depicts, from left to right, thesequence of eluted material from the cHA column. The Y-axis depicts bothUV absorbance (at 280 nM) and conductivity, as separately noted in thegraph. The Parent 1 and Parent 2 peaks are smaller in FIG. 5 than inFIG. 3, because the sample loaded on the cHA column for FIG. 5 was notspiked with additional Parent 1 and Parent 2 antibody (whereas for FIG.3, the sample was spiked with additional Parent 1 and Parent 2antibody).

FIG. 6 provides a graph showing additional information about recovery ofthe intact anti-BCMA/CD3 bispecific antibody of interest from the cHAresin, as well as the relative amount of clipped bispecific antibody invarious eluted fractions from the cHA resin. The X-axis depicts, fromleft to right, the sequence of eluate from the cHA column (i.e. from lowsalt to high salt; each data point indicates a fraction eluted from thecolumn). Along the vertical/Y-axis, three different variables areplotted for each fraction: Variable 1)(diamonds): the cumulative intactbispecific antibody recovery (“% P01”), which is the total amount ofintact anti-BCMA/CD3 bispecific antibody (i.e. the protein of interest)recovered from the cHA column through that fraction in thechromatography run (put another way, it is as if all of material elutedfrom the cHA column during the run up to and including that fraction arecollected and pooled, and then the total amount of intact anti-BCMA/CD3bispecific antibody in that pooled material is measured); also, the “%POI” value is presented as the percentage of the total amount of theintact anti-BCMA/CD3 bispecific antibody/protein of interest that wasloaded onto the cHA column which has been recovered (i.e. rather thanbeing presented as a value in grams). Variable 2)(squares): the % of theprotein in each respective fraction which is the clipped bispecificantibody (“% Clip”). Variable 3)(triangles): the cumulative clippedbispecific antibody recovery (“Cumulative Clip”), which is the totalamount of clipped bispecific antibody recovered from the cHA columnthrough that fraction in the chromatography run. In addition, in theFIG. 6 graph, the left side Y-axis lists values for the % POI, and theright side Y-axis lists values for % Clip.

FIG. 6 also contains information showing how certain different fractionsin FIG. 6 correspond to different points in the UV absorbance in thechromatography profile as shown in FIG. 5. So, for example, FIG. 5 showsthat the largest peak of UV absorbance/protein elution from the cHAresin corresponds to the protein of interest/intact anti-BCMA/CD3bispecific antibody. The top of this large peak of UV absorbance/proteinelution from the cHA column is also referred to as the “Apex” of therun; the chromatography fraction peak corresponding to this Apex pointis noted on FIG. 6. Then, various points after the Apex peak are alsonotated in FIG. 6. These points are “90%”, “80%”, “70%”, “60%”, and“50%”, and they are calculated as follows. The UV absorbance value atthe Apex is set as the starting point for additional calculations. Then,the UV value which is 90% of the Apex UV value is determined. The 90%Apex UV value which occurs after the Apex UV value is achieved duringthe chromatography run (i.e. towards the tail end of the peak) is notedas the “90%” fraction; it may also be referred to herein as the “90%post Post Apex” fraction, or the like. This process is repeated for the80%, 70%, 60%, and 50% values (i.e. each of these values is further downthe tail end from the peak, and thus represents an increasingly largeamount of collected material). As FIG. 6 shows, as the % of the Apexvalue decreases, the % Clip in the fractions increases. This isconsistent with the process in which the clipped bispecific antibodyelutes from the cHA column at a later time/under higher salt conditionsthan the intact anti-BCMA/CD3 bispecific antibody. Accordingly, if alarger fraction of the Apex peak is collected, (i.e. to a lower % postPeak Apex fraction), then more clipped bispecific antibody will also becollected, due to the partial overlap between the elution profiles ofthe intact bispecific antibody and the clipped bispecific antibody.

Various values from FIG. 6 are also provided below in Table 1. As shownin Table 1, according to the cHA purification method provided herein,for example, if the 90% post Peak Apex eluate from the cHA column iscollected (i.e. the POI % recovery through the “90% post Peak Apex”fraction), then 54% of the intact bispecific antibody/protein ofinterest that was loaded onto the cHA column is recovered, and thisrecovered protein pool contains 0% clipped bispecific antibody. Inanother example, if the 50% post Peak Apex eluate from the cHA column iscollected (i.e. the POI % recovery through the “50% post Peak Apex”fraction), then 74% of the intact bispecific antibody/protein ofinterest that was loaded onto the cHA column is recovered, and thisrecovered protein pool contains 0.2% clipped bispecific antibody.Accordingly, as shown in FIG. 6 and Table 1, robust purification of theintact bispecific antibody of interest from the clipped bispecificantibody can be effectively achieved via a cHA resin.

TABLE 1 Peak Pool % POI % Cumulative Clip Peak Apex 34 0 90% post PeakApex 54 0 80% post Peak Apex 61 0.1 70% post Peak Apex 67 0.1 60% postPeak Apex 70 0.2 50% post Peak Apex 74 0.2

For the cHA chromatography results as shown in FIGS. 3-6, the cHAchromatography was performed as follows. The antibody preparationcontaining the bispecific anti-BCMA/CD3 antibody of interest and variousimpurities was loaded onto a chromatography column containing a cHAresin. The cHA resin was cHA Type 1, 40 μM bead size (Bio-Rad). For thechromatography runs depicted in FIGS. 3 and 4, the cHA resin was loadedwith sample to a protein density on the cHA resin of 30 g/L. For thechromatography runs depicted in FIGS. 5 and 6, the cHA resin was loadedwith sample to a protein density on the cHA resin of 10 g/L. Beforeloading sample onto the cHA resin, the resin was pre-equilibrated with 5column volumes of Equilibration Buffer 1, followed by 5 column volumesof Equilibration Buffer 2. The composition of the various buffersdescribed in this method are listed below in Table 2. The antibodypreparation containing the partially purified bispecific anti-BCMA/CD3antibody of interest was loaded onto the cHA resin column in a LoadBuffer containing approximately 50 mM Tris and 60 mM glycine, pH 7.5.After the antibody preparation was loaded onto the cHA resin column, thecolumn was then washed with 3 column volumes of Wash Buffer. Then, thebispecific antibody of interest (i.e. the intact bispecific antibody)was eluted from the cHA resin using a 20 column volumes of ElutionBuffer, in which the sodium phosphate concentration in the ElutionBuffer was increased from 40 mM to 80 mM over the course of the elution.As shown in FIGS. 3-6, the various impurities that were present in theantibody preparation with the bispecific antibody of interest also elutefrom the cHA column in response to the Elution Buffer, but they do sounder sufficiently different salt concentrations from the intactanti-BCMA/CD3 bispecific antibody of interest, such that the intactbispecific antibody can be effectively separated from the variousimpurities, including the clipped version of the intact bispecificantibody.

After the protein of interest is eluted from the cHA resin column by theElution Buffer according to the protocol described above, the cHA resinmay then be stripped with 5 column volumes Strip Buffer, followed bysanitization with 5 column volumes Sanitization Buffer, followed by 5column volumes of Storage Buffer.

For each of the above analyses of different material eluted from the cHAcolumn, the type and amount of the different molecular species invarious fractions/pools was determined by analytical cation exchange(CEX) analysis.

TABLE 2 Buffer Name Composition Column Volume(s) Equilibration 400 mMsodium phosphate, 5 Buffer 1 pH 7.5 Equilibration 20 mM HEPES, 2 mM 5Buffer 2 sodium phosphate, pH 7.5 Load Buffer Protein pool in N/Aapproximately 50 mM Tris, 60 mM glycine, adjusted to pH 7.5; Wash Buffer20 mM HEPES, 40 mM 3 sodium phosphate Elution Buffer 20 column volumegradient 20 from 40-100 mM sodium phosphate Strip Buffer 400 mM sodiumphosphate, 5 pH 7.5 Sanitization 500 mM potassium 5 Buffer phosphate, 1Msodium hydroxide Storage Buffer 100 mM sodium hydroxide 5

Example 2: Purification of an Anti-BCMA/Anti-CD3 Bispecific Antibody bycHA Resin Chromatography—Mass Loading Challenges Objective:

The objective in this example was to determine whether the bispecificantibody purification method described in Example 1 could be effectivelyused with various cHA resin column mass loading challenges. For example,a specific objective was to determine whether, for various mass loadingchallenges on the cHA resin column, it would be possible to consistentlyrecover more than 50% of the input bispecific antibody, while at thesame time having no more than 1% clipped bispecific antibody as impurityin the recovered, purified bispecific antibody product.

Materials and Methods:

The materials and methods for this Example were the same as in Example1, except that the cHA resin was loaded with antibody preparation sampleto a protein density on the cHA resin (in different chromatography runs)of 8 g/L, 10 g/L, or 12 g/L.

Results:

The results from these chromatography runs are summarized below in Table3. For all 3 of the chromatography runs, the 90% post Peak Apex material(as described in Example 1) was collected and analyzed. As shown inTable 3, for each of the 8 g/L, 10 g/L, and 12 g/L mass challenges, over50% of the loaded intact bispecific antibody was recovered as purifiedbispecific antibody, and the recovered purified bispecific antibodyproduct contained less than 1% clipped bispecific antibody as animpurity.

Thus, these experiments show that the intact bispecific antibody ofinterest can be consistently effectively separated from the clippedbispecific antibody by cHA resin chromatography, at various cHA resinmass loading challenges.

TABLE 3 Mass Challenge Cumulative POI Recovery Cumulative Clip  8grams/liter 56% 0.4 10 grams/liter 58% 0.5 12 grams/liter 58% 0.8

Example 3: Purification of an Anti-BCMA/Anti-CD3 Bispecific Antibody bycHA Resin Chromatography—Different pH Challenges Objective:

The objective in this example was to determine whether the bispecificantibody purification method described in Example 1 could be could beeffectively performed under different pH conditions.

Materials and Methods:

The materials and methods for this Example were the same as in Example1, except that the pH of the buffers used throughout the method was pH7.0, pH 7.5, or pH 8.0 (in different chromatography runs). For thesechromatography runs, the cHA resin was loaded with antibody preparationsample to a protein density of 30 g/L on the cHA resin.

Results:

The results from these chromatography runs are summarized below in Table4. For all 3 of the chromatography runs, the Peak Apex material (asdescribed in Example 1) was collected and analyzed. As shown in Table 4,the intact bispecific antibody protein of interest was successfullyrecovered for each of the different tested pH conditions, and pH 7.5yielded the highest recovery of the protein of interest. (The amount ofclipped bispecific antibody in the purified material was not separatelydetermined for these chromatography runs.)

Thus, these experiments show that the intact bispecific antibody ofinterest can be effectively purified by cHA resin chromatography atdifferent pH conditions.

TABLE 4 Buffer pH Cumulative POI Recovery 7.0 30% 7.5 40% 8.0 20%

Example 4: Purification of an Anti-FLT3/Anti-CD3 Bispecific Antibody bycHA Resin Chromatography Objective:

The objective of this example was to determine whether the bispecificantibody purification method described in Example 1 could be effectivelyperformed with a different bispecific antibody than used in Example 1,in which there also was a need to separate an intact bispecific antibodyof interest from various impurities, including a related clippedbispecific antibody of similar mass. In this example, the antibody ofinterest was a heterodimeric bispecific anti-FLT3/anti-CD3 antibody.

Materials and Methods:

The heterodimeric bispecific anti-FLT3/anti-CD3 antibody used in thisexample contained the same anti-CD3 heavy chain and light chain as inExample 1. The amino acid sequences of the polypeptides in the FLT3 armof the bispecific antibody are shown in the following SEQ ID NOs: FLT3heavy chain: SEQ ID NO: 10; FLT3 light chain: SEQ ID NO: 12.

The clipped version of the bispecific antibody in the example had a clipin the same position of the anti-CD3 heavy chain as described in Example1.

The materials and methods for this Example were the same as in Example1, except that that, as described above, the antibody preparation samplecontained bispecific anti-FLT3/anti-CD3 antibody. The cHA resin wasloaded with antibody preparation sample to protein density on the cHAresin of 10 g/L.

Results:

FIG. 7 shows a graph that provides information about the recovery of theintact bispecific anti-FLT3/CD3 antibody of interest from the cHA resin,as well as the relative amount of clipped bispecific antibody in variouseluted fractions from the cHA resin. The X-axis depicts, from left toright, the sequence of eluate from the cHA column (i.e. from low salt tohigh salt; each data point indicates a fraction eluted from the column).Along the vertical/Y-axis, three different variables are plotted foreach fraction: Variable 1)(diamonds): % P01; Variable 2)(squares): %Clip; and Variable 3)(triangles), each of which was determined asdescribed in Example 1 for FIG. 6. In addition, in the FIG. 7 graph, theleft side Y-axis lists values for the % POI, and the right side Y-axislists values for % Clip. FIG. 7 also contains information showing howcertain different fractions in FIG. 7 correspond to different points inthe UV absorbance in the corresponding chromatography profile (notshown). The points notated as “Apex”, “85%”, and “60%” were determinedin the same way as described for FIG. 6.

Various values from FIG. 7 are also provided below in Table 5. As shownin Table 5, the cHA resin purification method provided herein permits,for example, the recovery of over 80% intact bispecific anti-FLT3/CD3antibody of interest, while having less than 1% clipped bispecificantibody in the purified antibody product.

Accordingly, as shown in the FIG. 7 and Table 5, purification of intactbispecific anti-FLT3/CD3 from the clipped bispecific antibody can beeffectively achieved via a cHA resin.

TABLE 5 Peak Pool % POI % Cumulative Clip Peak Apex 55 0.2 85% post PeakApex 72 0.4 60% post Peak Apex 82 0.7

Although the disclosed teachings have been described with reference tovarious applications, methods, kits, and compositions, it will beappreciated that various changes and modifications can be made withoutdeparting from the teachings herein and the claimed invention below. Theforegoing examples are provided to better illustrate the disclosedteachings and are not intended to limit the scope of the teachingspresented herein. While the present teachings have been described interms of these exemplary embodiments, the skilled artisan will readilyunderstand that numerous variations and modifications of these exemplaryembodiments are possible without undue experimentation. All suchvariations and modifications are within the scope of the currentteachings.

All references cited herein, including patents, patent applications,papers, text books, and the like, and the references cited therein, tothe extent that they are not already, are hereby incorporated byreference in their entirety. In the event that one or more of theincorporated literature and similar materials differs from orcontradicts this application, including but not limited to definedterms, term usage, described techniques, or the like, this applicationcontrols.

The foregoing description and Examples detail certain specificembodiments of the invention and describes the best mode contemplated bythe inventors. It will be appreciated, however, that no matter howdetailed the foregoing may appear in text, the invention may bepracticed in many ways and the invention should be construed inaccordance with the appended claims and any equivalents thereof.

It is understood that wherever embodiments are described herein with thelanguage “comprising,” otherwise analogous embodiments described interms of “consisting of” and/or “consisting essentially of” are alsoprovided.

Where aspects or embodiments of the invention are described in terms ofa Markush group or other grouping of alternatives, the present inventionencompasses not only the entire group listed as a whole, but each memberof the group individually and all possible subgroups of the main group,but also the main group absent one or more of the group members. Thepresent invention also envisages the explicit exclusion of one or moreof any of the group members in the claimed invention.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. In case of conflict, thepresent specification, including definitions, will control. Throughoutthis specification and claims, the word “comprise,” or variations suchas “comprises” or “comprising” will be understood to imply the inclusionof a stated integer or group of integers but not the exclusion of anyother integer or group of integers. Unless otherwise required bycontext, singular terms shall include pluralities and plural terms shallinclude the singular. Any example(s) following the term “e.g.” or “forexample” is not meant to be exhaustive or limiting. The term “or” whenused in the context of a listing of multiple options (e.g. “A, B, or C”)shall be interpreted to include any one or more of the options, unlessthe context clearly dictates otherwise.

Exemplary methods and materials are described herein, although methodsand materials similar or equivalent to those described herein can alsobe used in the practice or testing of the present invention. Thematerials, methods, and examples are illustrative only and not intendedto be limiting.

1. A method of purifying an antibody comprising: A) loading an antibodypreparation in a load buffer onto a hydroxyapatite (HA) resin, wherein:the antibody preparation comprises: I) an intact antibody of interestand II) a clipped version of the antibody of interest, wherein theclipped version of the antibody of interest is a degradation productionfrom the intact antibody of interest, and has a mass that is less than10% different than the mass of the intact antibody of interest; and B)eluting the intact antibody of interest from the HA resin with anelution buffer comprising an ion, wherein the concentration of the ionin the elution buffer is increased during the elution.
 2. A method ofpurifying a bispecific antibody comprising: A) loading an antibodypreparation in a load buffer onto a hydroxyapatite (HA) resin, wherein:the antibody preparation comprises: I) an intact bispecific antibody ofinterest; and II) at least one impurity species, wherein the impurityspecies are selected from the group consisting of: a) a clipped versionof the bispecific antibody of interest, wherein the clipped version ofthe bispecific antibody of interest is a degradation production from theintact bispecific antibody of interest, and has a mass that is less than10% different than the mass of the intact bispecific antibody ofinterest; b) a first parent antibody, wherein the first parent antibodyis a monospecific antibody having the same antigen specificity as afirst arm of the intact bispecific antibody; c) a second parentantibody, wherein the second parent antibody is a monospecific antibodyhaving the same antigen specificity as a second arm of the intactbispecific antibody; and d) high molecular mass species (HMMS); and B)eluting the intact bispecific antibody of interest from the HA resinwith an elution buffer comprising an ion, wherein the concentration ofthe ion in the elution buffer is increased during the elution.
 3. Amethod of purifying a bispecific antibody comprising: A) loading anantibody preparation in a load buffer onto a hydroxyapatite (HA) resin,wherein: I) the antibody preparation comprises: a) an intact bispecificantibody of interest and b) a clipped version of the bispecific antibodyof interest, wherein the clipped version of the antibody of interest isa degradation production from the intact bispecific antibody ofinterest, and has a mass that is less than 10% different than the massof the intact bispecific antibody of interest; and II) the ratio ofmolecules of the clipped bispecific antibody to molecules of the intactbispecific antibody in the antibody preparation is between is between atleast 1:50 and no greater than 1:5; B) eluting the intact bispecificantibody from the HA resin with an elution buffer comprising an ion,wherein the concentration of the ion in the elution buffer is increasedduring the elution, and optionally, C) collecting a purified fractioneluted from the HA resin, wherein the purified fraction comprises theintact bispecific antibody.
 4. The method of claim 1, wherein theantibody is a heterodimeric bispecific antibody.
 5. The method of claim1, further comprising collecting a purified fraction eluted from the HAresin, wherein the purified fraction comprises the intact antibody ofinterest, and wherein the purified fraction comprises at least 95%, 96%,97%, 98%, or 99% by mass intact antibody of interest.
 6. The method ofclaim 3, wherein the purified fraction comprises the intact bispecificantibody and the clipped bispecific antibody, further wherein the ratioof clipped bispecific antibody molecules to intact bispecific antibodymolecules in the purified fraction is no greater than 1:100.
 7. Themethod of claim 1, wherein the antibody of interest is an anti-CD3antibody, and wherein the antibody comprises at least one of thefollowing: i) a VH region comprising an amino acid sequence as shown inSEQ ID NO: 1; ii) a heavy chain comprising an amino acid sequence asshown in SEQ ID NO: 2; iii) a VH region comprising an amino acidsequence as shown in SEQ ID NO: 1 and a VL region comprising an aminoacid sequence as shown in SEQ ID NO: 3; or iv) a heavy chain comprisingan amino acid sequence as shown in SEQ ID NO: 2 and a light chaincomprising an amino acid sequence as shown in SEQ ID NO:
 4. 8. Themethod of claim 2, wherein the bispecific antibody is: i) ananti-BCMA/anti-CD3 bispecific antibody comprising an anti-BCMA arm andan anti-CD3 arm, or ii) an anti-FLT3/anti-CD3 bispecific antibodycomprising an anti-FLT3 arm and an anti-CD3 arm.
 9. The method of claim8, wherein the anti-CD3 arm comprises at least one of the following: i)a VH region comprising an amino acid sequence as shown in SEQ ID NO: 1;ii) a heavy chain comprising an amino acid sequence as shown in SEQ IDNO: 2; iii) a VH region comprising an amino acid sequence as shown inSEQ ID NO: 1 and a VL region comprising an amino acid sequence as shownin SEQ ID NO: 3; or iv) a heavy chain comprising an amino acid sequenceas shown in SEQ ID NO: 2 and a light chain comprising an amino acidsequence as shown in SEQ ID NO:
 4. 10. The method of claim 8, whereinthe anti-BCMA arm comprises at least one of the following: i) a VHregion comprising an amino acid sequence as shown in SEQ ID NO: 5; ii) aheavy chain comprising an amino acid sequence as shown in SEQ ID NO: 6;iii) a VH region comprising an amino acid sequence as shown in SEQ IDNO: 5 and a VL region comprising an amino acid sequence as shown in SEQID NO: 7; or iv) a heavy chain comprising an amino acid sequence asshown in SEQ ID NO: 6 and a light chain comprising an amino acidsequence as shown in SEQ ID NO:
 8. 11. The method of claim 8, whereinthe anti-FLT3 arm comprises at least one of the following: i) a VHregion comprising an amino acid sequence as shown in SEQ ID NO: 9; ii) aheavy chain comprising an amino acid sequence as shown in SEQ ID NO: 10;iii) a VH region comprising an amino acid sequence as shown in SEQ IDNO: 9 and a VL region comprising an amino acid sequence as shown in SEQID NO: 11; or iv) a heavy chain comprising an amino acid sequence asshown in SEQ ID NO: 10 and a light chain comprising an amino acidsequence as shown in SEQ ID NO:
 12. 12. The method of claim 1, whereinthe antibody preparation is loaded onto the HA resin to a density on theresin of between 5 g/L and 20 g/L.
 13. The method of claim 1, wherein atleast 1 gram of antibody preparation is loaded onto the HA resin. 14.The method of claim 1, wherein the antibody preparation comprises atleast 50% but less than 95% by mass intact antibody of interest.
 15. Themethod of claim 1, wherein the clipped antibody has a mass that lessthan 1% different than the mass of the intact antibody.
 16. The methodof claim 1, wherein the clipped antibody has a mass that is betweenabout 5 and 100 Daltons greater than the mass of the intact antibody.17. The method of claim 1, wherein the clipped antibody has a cleavedpeptide bond in a polypeptide chain of the antibody, and wherein thecleaved peptide bond is in a heavy chain of the antibody.
 18. The methodof claim 1, wherein the clipped antibody contains the same number ofamino acids and the same amino acid sequences as the intact antibody.19. The method of claim 1, wherein the clipped antibody contains adifferent number of amino acids as the intact antibody.
 20. The methodof claim 1, wherein the antibody of interest comprises a VH and VLdomain which specifically bind to CD3, and wherein the clipped antibodycomprises a cleaved peptide bond in the VH domain that specificallybinds CD3.
 21. The method of claim 1, wherein the HA resin is ceramichydroxyapatite (cHA) resin.
 22. The method of claim 1, wherein afterloading the antibody preparation onto the HA resin but prior to elutingthe intact bispecific antibody the resin is washed with a wash buffercomprising phosphate ions at concentration between 10 and 50 mM.
 23. Themethod of claim 1, wherein the ion in the elution buffer is phosphate.24. The method of claim 1, wherein the concentration of the phosphateion is increased during the elution from about 40 mM to 100 mM.
 25. Themethod of claim 1, wherein the pH of at least one of the load buffer,wash buffer, and elution buffer is at or between about pH 7.0 and 8.0.26. The method of claim 1, wherein the antibody preparation containsproteins that were previously loaded onto and eluted from at least oneof: i) a protein A resin and ii) an ion exchange resin.
 27. The methodof claim 1, wherein the antibody is isolated and/or purified for use asor in the preparation of pharmaceuticals.